Chemotherapy drugs induce CD147 internalization

Our analysis of two sets of differentially expressed RNA sequencing genes resistant and sensitive to oxaliplatin in the GEO database and enrichment of KEGG pathways showed upregulated genes enriched in pathways such as chemical carcinogenesis-reactive oxygen species pathway, cell adhesion molecules pathway, Efferocytosis pathway, phagosome pathway, non-alcoholic fatty liver disease pathway, lysosome pathway, and so on (Fig. 1A-C). Based on lysosomal pathways associated with autophagy, we screened 63 genes with differential expression of more than two-fold (Fig. 1D). We selected lysosomal tripeptidyl peptidase (TPP1) and CD147 (BSG) based on their protein degree value and betweenness centrality calculated using the topological network analysis software Cytoscape (Fig. 1E). In TCGA-LICH cancer and normal liver tissues, as well as GTEx, we found a significant correlation between TPP1 and CD147 (R = 0.46, Fig. 1F). In previous studies, CD147 could internalize and target lysosomes [18]. We speculate that clinical chemotherapy resistance is related to CD147 internalization targeting the lysosome pathway based on results of bioinformatics analysis. Hence, we verified that GFP-labeled CD147 co-localized with lysosomes in human embryonic kidney (HEK) HEK293T cells treated with treated with lysosomal inhibitor chloroquine (CQ) (20 μM) for 12 h (Fig. 1G, S1B). Cisplatin (DDP) was applied in different doses (0 μg / mL, 5 μg / mL, and 10 μg / mL) for 12 h in 293 T cells to investigate the internalization effect of platinum-based chemotherapy drugs on CD147. Interestingly, super-resolution images and living-cell images both showed that CD147 internalizes targeted lysosomes, and with increasing concentration, more green fluorescence occured in lysosomes (P = 0.0094; Fig. 1H, I; Movie 1). There was a significant difference in the co-localization of CD147 and lysosomes compared to the control group in 5 μg / mL and 10 μg / mL DDP (P = 0.0312, P < 0.0001; Figures S1C, D), suggesting that chemotherapy drugs could induce CD147 internalization.

Fig. 1

Chemotherapy drugs induce CD147 internalization. A RNA-seq principal component analysis of liver cancer tissue samples with and without oxaliplatin resistance. B A heat map of differentially expressed genes in liver cancer tissues with oxaliplatin resistance and response, adjusted P value < 0.05. C A KEGG enrichment analysis of differentially expressed genes in liver cancer tissues with oxaliplatin resistance and response. D Differential gene heatmap enriched in the KEGG Lysome pathway, with Fold Change > 2, adjusted P value < 0.05. E PPI protein mutual network is established by screening core proteins. F mRNA level correlation between TPP1 (lysosomal tripeptidyl peptidase) and BSG (CD147). G Laser confocal microscopy images of HEK293T cells treated with chloroquine (CQ) (20 μM) for 12 h. Scale bar: 20 μm. H Elyra 7 microscope images of 293 T cells transfected with eGFP-CD147(N) and stained with lysotracker. Scale bar: 10 μm. I Quantitation of green average fluorescence intensity in co-localization spots with Zeiss ZEN 3.4 (blue edition) software. Data are the means ± SD of three images in each group. Statistical significance by unpaired, two-tailed Student’s t-test; **, P < 0.01

CD147 intracellular segments internalized and localized on lysosomes

In previous studies, low cholesterol inhibitors have been shown to promote hydrolytic cleavage of the extracellular segment of CD147. Once the inhibitor was removed, the intracellular segment of CD147 could be internalized into the nucleus [18]. To observe this internalization phenomenon, we transfected the CD147-enhanced green fluorescent protein (eGFP) plasmid, in which GFP was fused at the C-terminus, into HEK293T cells and observed the localization of CD147 residual segments using laser scanning confocal microscopy. Following the withdrawal of methyl-β-cyclodextrin (MβCD) treatment, CD147 fluorescence on the cell membrane weakened, internalized, and diffused into the cytoplasm (Fig. 2A). Here, the expression of CD147-marked intracellular segments on the cell membrane was detected by flow cytometry (FC) in Huh7 (P = 0.0459, P = 0.0354, P = 0.088, respectively) and HCCLM3 (P < 0.0001, P = 0.0006, P = 0.0012, respectively). CD147 expression decreased on the cell membrane compared to that in the control group after treatment with the cholesterol inhibitor (Fig. 2B-E), and 10 mM concentration of MβCD resulted in a more obvious shedding effect on the extracellular segment of CD147 (Fig. 2C, E). The HAb18 monoclonal antibody was used to detect CD147 extracellular segment expression of Huh7 or HCCLM3 cells. Consistent with FC results, the expression of CD147 extracellular segments was significantly lower than that in the control group (P = 0.0141, P = 0.0295; Fig. 2F, G), which confirmed that low cholesterol levels enhanced the shedding of the extracellular segments and the internalization of intracellular segments of CD147. Simultaneously, 3D images and living cell images also showed significant co-localization between internalized CD147 intracellular domains and lysosomes, with localization distributed near nucleus (Fig. 2H, Movie 2, Figures S1A, 2C). These results confirmed that Low-concentration cholesterol contributed to the shedding of the extracellular segments of CD147 and the internalization of the intracellular domain of residual CD147.

Fig. 2

CD147 intracellular segments internalized and localized on lysosomes. A Images of HEK293T cells that were treated with MβCD (10 mM) for two hours and then the MBCD treatment was removed. Scale bar: 20 μm. B, D In Huh7 and HCCLM3 cells, CD147 was labelled with FITC (BD, 555962). After treating cells with different concentrations of MBCD (0, 5, 10, and 15 mM) for two hours, the MBCD treatment was removed, and then flow cytometry was used to detect the expression of CD147 on the cell membrane. C, E A quantitative analysis is performed on CD147 extracellular segment expression in B and D. Data are means ± SD of three biological replicates. Statistical significance by unpaired, two-tailed Student’s t-test; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. F Western blotting was used to detect the expression of the CD147 extracellular segment. G Analysis of CD147 extracellular segment expression in F. Data are means ± SD of three biological replicates. H Micrographs taken with the Elyra 7 microscope of HEK293T cells which were treated with MβCD (10 mM) for two hours, the MBCD treatment was removed. Scale bar: 10 μm. I Schematic diagram (drawn using the Integrated Business Services software) depicting the fusion mode of GFP and CD147. J Western blotting analysis of the intracellular domain of residual CD147 in Huh7 and HepG2 cells. K Laser confocal microscopy results of Huh7 CtrlGFP and Huh7 △ECD-GFP cells; LAMP1 (Santa Cruz, sc-20011)-labeled lysosomes; DAPI-labeled nuclei. Scale bar: 10 μm; 20 μm. L Laser confocal microscopy results of HepG2 CtrlGFP and HepG2 △ECD-GFP cells; LAMP1-labeled lysosomes; DAPI-labeled nuclei. Scale bar: 5 μm; 20 μm. M Laser confocal microscopy results of Huh7 CtrlGFP and Huh7 △ECD-GFP cells treated with chloroquine (CQ); LAMP1-labeled lysosomes; DAPI-labeled nuclei. Scale bar: 5 μm; 20 μm. N Colocalization images of CD147 and lysosomes. Pearson's colocalization coefficient (PCC) was used to quantify the degree of colocalization

To investigate the localization of the intracellular domain of residual CD147 followed by hydrolytic cleavage of the extracellular segments in hepatoma cells, we transfected lentiviral overexpressing the transmembrane and intracellular domains of CD147 (△ECD-GFP) into Huh7 and HepG2 cells and screened the stably transfected cell lines for puromycin resistance (Fig. 2I). △ECD-GFP was successfully transfected into hepatoma cells. A band of approximately 34 kDa was detected in both cell lines by western blotting using the CD147 C-terminal antibody (Fig. 2J). We found that the intracellular domain of residual CD147 co-localized with lysosomes in Huh7 and HepG2 cells (Fig. 2K, L, and N). After treatment with CQ, the co-localization of CD147 intracellular segments and lysosomes increased (Fig. 2M, N). The plot profile tool in Image J was used to analyze the coincidence of CD147 intracellular segments and lysosome-associated membrane protein 1 (LAMP1) at the same spatial position. The overlapping area of the red broken line and green broken line significantly increased after adding the lysosomal inhibitor CQ to Huh7 cells (Figure S2E). Immunoelectron microscopy clearly showed that the intracellular fragments of CD147 were not only located on the cytoplasmic side of the membrane in Huh7 CtrlGFP and Huh7 △ECD-GFP but were located on the surface of lysosomes in Huh7 △ECD-GFP (Figures S2F, G). These results confirmed the internalization of the intracellular domain of residual CD147, which was co-located with lysosomes after the shedding of the extracellular segments of CD147.

CD147 intracellular segments formed a complex with G3BP1 on lysosomes

To further confirm the localization of CD147 internalization on the surface of lysosomes, we extracted lysosomes from cells. The absence of expression of the Golgi membrane (GM) proteins GM130, and Lamin B1 indicated the high purity of the extracted lysosomes by column centrifugation (Figure S2A). Intracellular segments of CD147 were also identified in the total lysosomal protein extracted by mass spectrometry (MS) (Fig. 3A, S2D). The fluorescence of CD147 intracellular segments on the lysosomal membrane was detected using FC. Results showed that the fluorescence of the intracellular segments of CD147 in Huh7 △ECD-GFP was stronger than that in the control group (Fig. 3B, S2B), consistently, the ratio of CD147 positive lysosomes in Huh7 △ECD-GFP was significantly higher than that in the control group (P = 0.0126; Fig. 3C). Although the intracellular domain of residual CD147 internalizes induced by DDP, how the intracellular domain of residual CD147 is transferred from the cell membrane to lysosomes remains unknown. Proteins interacting with the intracellular domain of residual CD147 in lysosomes were detected by CO-IP and MS, and G3BP1 and Rab7A were screened (Table S1). G3BP1 is a core molecule usually located in stress granules (SGs) [24]. Rab7A, a member of the Rab family of small G proteins, regulates the translocation of early endosomes to late endosomes, and late endosomes to lysosomes [25]. Immunoblotting verified that CD147 intracellular segments interacted with G3BP1 and Rab7A (Fig. 3D, S3C). It was reported that inhibition of lysosomal function affected the transport of compound targets to lysosomes by Rab7A [26]. After CQ addition, the colocalization of △ECD-GFP and Rab 7A on lysosomes disappeared in Huh7 cells (Figure S3B). It was confirmed that the purified G3BP1 protein with His tag directly binds to the synthesized △ECD-GFP with GST tag by GST pulldown (Fig. 3E). Laser confocal images showed that overexpressed CD147 intracellular segments co-localized with G3BP1 in Huh7△ECD-GFP cells (Fig. 3F). However, the co-localization of G3BP1 and lysosomes appeared only when CD147 was overexpressed (Figure S3A). Immunoelectron microscopy images also showed that the intracellular domains of residual CD147 and G3BP1 were co-localized near lysosomes in Huh7△ECD-GFP cells (P = 0.0188, P = 0.0413, P = 0.0031; Fig. 3K-M). To verify that the combination was due to internalization of the CD147 intracellular domain, we transfected pmCherry-ΔECD and pGFP-G3BP1 into HEK293T cells and performed immunofluorescence staining of Rab7A. Laser confocal images showed that mCherry-△ECD, mCherry-ICD, GFP-G3BP1, and Rab7A co-localized in lysosomes respectively (Fig. 3O, S3D, and S3E). However, co-localization of GFP-G3BP1 and Rab7A was not observed in the absence of CD147 intracellular segments (Figure S3F). In order to further detect the morphology of CD147 intracellular segments that are bound to G3BP1, we transfected pmCherry-G3BP1 into Huh7 CtrlGFP and Huh7 △ECD-GFP. Super-resolution images showed that overexpressed CD147 intracellular segments colocalized with G3BP1 in similar vesicles compared with Huh7 CtrlGFP (Fig. 3N). Moreover, SPR results showed consistent results that CD147 △ECD directly binds to G3BP1 and the constant of combination was 3.20 × 10–8 mol/L (Fig. 3G-J). These results show that the intracellular domain of residual CD147 recruited G3BP1 after internalization that targeted lysosomes via Rab7A.

Fig. 3

CD147 intracellular segments formed a complex with G3BP1 on lysosomes. A Lysosomes were extracted from Huh7 cells using the lysosome extraction kit (Invent Biotechnologies, Inc. Eden Prairie, America) and analyzed by LC/LC–MS. B An FC analysis of CD147 intracellular segments on the lysosomal membrane was performed using LAMP1-PE (Santa Cruz, sc-19992-PE). C Data are the means and standard deviations of three biological replicates. Statistical significance is determined by an unpaired, two-tailed Student's t-test. D Western blot analysis of G3BP1 CO-IP with CD147 (Mouse IgG); CQ-stimulated Huh7 CtrlGFP and Huh7 △ECD-GFP cells (12 h). E The GST pull-down assay between the G3BP1 and CD147△ECD. F Results of laser confocal microscopy on Huh7 CtrlGFP cells and Huh7 △ECD-GFP cells; DAPI-labeled nuclei; Scale bar: 5 μm; 20 μm. G SPR curves of different IgG concentrations combined with G3BP1 as negative controls. H SPR curves of different CD147△ECD concentrations combined with G3BP1. I SPR curves of different G3BP1-antibody concentrations combined with G3BP1 as positive controls. J SPR curves of different CD147-antibody concentrations combined with CD147△ECD as positive controls. K Images of CD147 intracellular segments and G3BP1 immunoelectron microscopy. GOLD-10 nm nanoparticles-labeled G3BP1; GOLD-20 nm nanoparticle-labeled intracellular segments of CD147. Scale bar: 2 μm; 200 nm. L Quantifying intracellular distance between CD147 intracellular fragment and G3BP1 within the same cell. Data are the means ± SD of four images in each group. Statistical significance by unpaired, two-tailed Student’s t-test; *, P < 0.05. M Quantifying intracellular distance between CD147 intracellular fragment or G3BP1 and lysosomes within the same cell. Data are the means ± SD of six images in each group. Statistical significance by unpaired, two-tailed Student’s t-test; **, P < 0.01; *, P < 0.05. N Elyra 7 microscope images of Huh7 transfected with pmCherry-G3BP1. Scale bar: 10 μm. O Image of HEK293T cells transfected with pLVX-GFP-G3BP1 and pmCherry-△ECD; DAPI-labeled nuclei. Scale bar: 25 μm

CD147 intracellular segments enhanced cytoprotective autophagy via inhibited mTOR activity

mTORC1 protein plays a crucial role in autophagy regulation on lysosomes. Previous literature reported that G3BP1 can transform the migration ability of breast cancer cells by recruiting tuberous sclerosis complex (TSC) regulated the activity of mTORC1 in lysosomes [27]. To verify whether CD147-G3BP1 complex affects the activity of mTOR, we examined mTOR phosphorylation. The expression of phosphorylated mTOR (p-mTOR) at the Ser2448 site was significantly reduced in Huh7 △ECD-GFP cells compared to that in Huh7 CtrlGFP (P = 0.005; Fig. 4A, B). During autophagy, sequestosome-1 (SQSTM1) / p62 carries ubiquitinated proteins, binds to light chain 3 (LC3), is encapsulated by autophagosomes, and enters lysosomes for degradation. Western blotting results showed that the expression of SQSTM1/p62 was reduced (P = 0.015), whereas that of LC3B/A was significantly increased (P = 0.0082) in Huh7 △ECD-GFP cells compared to that in Huh7 CtrlGFP, whereas the expression of LC3B/A was significantly increased (P = 0.0006) in Huh7 △ECD-GFP cells treated with CQ (Fig. 4C, D). The expression of SQSTM1/p62 increased during obstructed autophagy because degradation was inhibited by CQ (P = 0.0406; Fig. 4C, D). Laser confocal microscopy also showed that the co-localization of LC3 and LAMP1 was more conspicuous in the experimental group treated with CQ than without CQ (Fig. 4E). In Huh7 △ECD-GFP cells, the coincidence area of LC3 (red) and lysosomes (green) was greater than that in the control group, as analyzed using the plot profile tool in ImageJ (Fig. 4F). To observe the autophagosome more accurately, we transferred the LC3 double fluorescent plasmid pCMV-mCherry-GFP-LC3B into Huh7 CtrlGFP and Huh7 △ECD-GFP cells. Normally, plasmids can simultaneously produce yellow fluorescence under excitation. Once LC3B was fused with the lysosome, green fluorescence was gradually quenched in the acidic environment of the lysosome, leaving only red fluorescence. After Hanks balanced salt solution (HBSS) starvation for four hours, it was found that there were more red fluorescent dots in Huh7 △ECD-GFP cells than in Huh7 CtrlGFP cells using Image J analysis (P = 0.0216; Fig. 4G, H). Immunoelectron microscopic images showed that the number of autolysosomes increased in Huh7 △ECD-GFP cells compared to that in Huh7 CtrlGFP (P = 0.0109), similarly, the number of autolysosomes also increased significantly treated with HBSS than without HBSS in Huh7 △ECD-GFP cells (P = 0.0083; Fig. 4I-K). The above results indicate that CD147 intracellular segments enhance autophagy in the hepatoma cells.

Fig. 4

CD147 intracellular segments enhanced autophagy flux via inhibited mTORC1 activity. A Results of Western blots for Huh7 wild type (WT), Huh7 CtrlGFP, and Huh7 △ECD-GFP. B Quantitation of p-mTOR expression in A. Data is the means ± SD of three biological replicates. Statistical significance by unpaired, two-tailed Student’s t-test; **, P < 0.01. C SQSTM1/p62 and LC3 expression in Huh7 WT, Huh7 CtrlGFP, and Huh7 △ECD-GFP cells. D Quantitation of SQSTM1/p62 and LC3 expression in C. Data is the means ± SD of three biological replicates. Statistical significance by unpaired, two-tailed Student’s t-test; **, P < 0.01; *, P < 0.05. E Laser confocal microscopy results of Huh7 CtrlGFP and Huh7 △ECD-GFP cells treated with CQ; LC3-labeled autophagosomes; LAMP1-labeled lysosomes; DAPI-labeled nuclei. Scale bar: 5 μm; 20 μm. F Image J plot profile tool used to locate LC3 and LAMP1 in the same spatial position. LAMP1 is indicated by the green broken line, and LC3 by the red broken line. G Images of Huh7 cells transfected with pmCherry-GFP-LC3B; HBSS-starved Huh7 CtrlGFP and Huh7 △ECD-GFP cells; DAPI-labeled nuclei. Scale bar: 5 μm; 20 μm. H Quantitation of the red immunofluorescence spot in G. Data are the means ± SD of three images in each group. Statistical significance by unpaired, two-tailed Student’s t-test; *, P < 0.05. I Quantitation of the number of autophagosome in J and K. Data are the means ± SD of six images in each group. Statistical significance by unpaired, two-tailed Student’s t-test; **, P < 0.01, *, P < 0.05. JK Transmission electron microscopy results of autophagy in Huh7 CtrlGFP and Huh7 △ECD-GFP. LD: lipid droplet; Mt: mitochondrion; Auto: autolysosome; Gn: glycogen; Scale bar: 5 μm; 1 μm

To explore what effect of autophagy induced by the intracellular domain of residual CD147, we treated Huh7 CtrlGFP and Huh7 △ECD-GFP cells with different concentrations of DDP (0 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL) for 48 h. The Cell counting kit-8 (CCK-8) assay showed that the sensitivity of Huh7△ECD-GFP cells to DDP was lower than that of Huh7 CtrlGFP (P = 0.0454; Figure S4A). The logIC50 value of Huh7 △ECD-GFP was significantly higher than that of Huh7 CtrlGFP (P = 0.0002; Figure S4B). We treated Huh7 CtrlGFP and Huh7 △ECD-GFP cells with DDP combined CQ. The CCK-8 assay showed that the sensitivity of Huh7 △ECD-GFP cells to DDP recovered to the same level as that of the control group (P = 0.0038; Figures S4A and B). It has been suggested that CD147 intracellular segments can induce cytoprotective autophagy. These results suggest that CD147 intracellular segments enhanced cytoprotective autophagy via inhibited mTOR activity.

G3BP1 knockout abated CD147-induced autophagy

To investigate the effect of intracellular internalization of full-length CD147 on autophagy in HCC cells, we overexpressed full-length CD147 with a FLAG tag (Fig. 5A). We detected autophagic flux and phosphorylated mTOR expression in cells at different time points after being treated with MβCD (Fig. 5B). We found that LC3B/LC3A expression gradually increased over time, especially the eighth hour after withdrawal of MβCD (P = 0.0104; Fig. 5C, D). Furthermore, p-mTOR expression significantly decreased, especially in sixth, eighth, and tenth hour (P = 0.0086, P = 0.0065, P = 0.0091, respectively; Fig. 5C, F). The period of mTOR activity inhibition coincided with the period when autophagic flux began to increase. However, SQSTM1/p62 expression fluctuated considerably, decreasing at the sixth hour (P = 0.0364) and increasing at the eighth hour (P = 0.0161), This might be because the change in SQSTM1/p62 always lagged behind LC3B (Fig. 5C-E). These results suggest that the internalization of endogenous CD147 intracellular segments can enhance autophagic flux by inhibiting mTOR activity in hepatoma cells.

Fig. 5

G3BP1 knockout abated CD147-induced autophagy. A Western blot analysis of CD147 in Huh7 wild type, Huh7 CtrlFlag, and Huh7 CD147Flag cells. B Schematic diagram of periodic administration and monitoring of cells. C Western blot analysis of p-mTOR, SQSTM1/p62, and LC3 at different time points after treatment with MβCD. D, E, F Quantitation of LC3, SQSTM1/p62, and p-mTOR expression. Data is the means ± SD of three biological replicates. Statistical significance by unpaired, two-tailed Student’s t-test; *, P < 0.05; **, P < 0.01. G Western blot results of CD147 in HCCLM3. H Western blot results of G3BP1in HCCLM3 △ECD-GFP cells transfected with sgNC, sgG3BP1 #1, sgG3BP1 #2, and sgG3BP1 #3. I Western blot results of G3BP1in HCCLM3 CtrlGFP cells transfected with sgNC, sgG3BP1 #1, sgG3BP1 #2, and sgG3BP1 #3. J Western blot results of p-mTOR in HCCLM3 CtrlGFP and HCCLM3 △ECD-GFP cells transfected with sgNC and sgG3BP1 #3. K Western blot results of SQSTM1/p62 and LC3 in HCCLM3 CtrlGFP and HCCLM3 △ECD-GFP cells transfected with sgNC and sgG3BP1 #3. L Western blot analysis of SQSTM1/p62 and LC3 in HCCLM3 CtrlGFP and HCCLM3 △ECD-GFP cells transfected with sgNC and sgG3BP1 #3 and treated with CQ. M Quantitation of p-mTOR expression. Data is the means ± SD of three biological replicates. Statistical significance by unpaired, two-tailed Student’s t-test; *, P < 0.05. N Quantitation of LC3B/LC3A expression. Data is the means ± SD of three biological replicates. Statistical significance by unpaired, two-tailed Student’s t-test; ***, P < 0.001; *, P < 0.05. O Quantitation of SQSTM1/p62 expression. Data is the means ± SD of three biological replicates. Statistical significance by unpaired, two-tailed Student’s t-test; *, P < 0.05

To verify the inhibitory effect of G3BP1 on mTORC1, we constructed HCC cell lines with high metastatic potential (HCCLM3) overexpressing the transmembrane and intracellular domains of CD147 and knocked down G3BP1 using three clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) single guide sequences (Table S3). We found that shG3BP1-3 had better interference efficiencies than shG3BP1-1 and shG3BP1-1 (Fig. 5H, I). We chose shG3BP1-3 for the follow-up experiments. We transfected shNC or shG3BP1-3 into HCCLM3 CtrlGFP and HCCLM3 △ECD-GFP cells. After G3BP1 knockout, the expression of p-mTOR significantly increased in HCCLM3 CtrlGFP and HCCLM3 △ECD-GFP (P = 0.0391 and P = 0.0371; Fig. 5J, M). LC3B/LC3A expression was significantly decreased in HCCLM3CtrlGFP and HCCLM3△ECD-GFP cells without and with CQ (P = 0.0142, P = 0.0472, P = 0.0008, P = 0.0189, respectively; Fig. 5K, L, and N). Moreover, SQSTM1/p62 expression was significantly reduced in HCCLM3△ECD-GFP cells without and with CQ (P = 0.0164, P = 0.0304; Fig. 5K, L, and O), but no significant difference was observed in HCCLM3 CtrlGFP. These results indicated that G3BP1 knockout abated CD147-induced autophagy in hepatoma cells.

Cisplatin-induced CD147 internalization formed a complex with G3BP1 accompanied by SGs depolymerization

Under chemotherapeutic stress, G3BP1 is activated to assemble stress granules (SGs) [28]. Laser confocal images showed that the number of SGs increased gradually in every 10 cells treated with an increased concentration of DDP in Huh7 CtrlGFP and Huh7 △ECD-GFP (P = 0.0142, P = 0.0063; Fig. 6A-C). However, compared with Huh7 CtrlGFP, the number of SG formed decreased at 2.5 μg / L and 5 μg / mL concentrations of DDP in Huh7 △ECD-GFP (P = 0.0062; Fig. 6C). To investigate whether CD147 intracellular segment overexpression was involved in SG formation under DDP, we transfected pmCherry-G3BP1 into HCCLM3 CtrlGFP and HCCLM3 △ECD-GFP which were observed by super-resolution microscopy. Super-resolution images showed that CD147 intracellular segments co-localized with G3BP1 at 0 μg / mL, 5 μg / mL and 10 μg / mL DDP in HCCLM3 △ECD-GFP, and the size of SGs assembled in △ECD-GFP bigger than CtrlGFP at 5 μg / mL DDP (Fig. 6D). To investigate whether CD147 affected SG depolymerization, we quantified the speckles area. The diameter of SG is usually greater than 100 nm. The images showed that as the concentration of DDP increased, the percentage of SGs gradually increased in HCCLM3 CtrlGFP and most of large volume SGs (area ≥ 5 μm2) appeared at 10 μg / mL DDP (Fig. 6D, E). However, compared to HCCLM3 CtrlGFP, large SGs of HCCLM3 △ECD-GFP appeared at 5 μg / mL DDP, depolymerized at 10 μg / mL DDP (Fig. 6D-G). For overexpressing CD147 intracellular segments, high-dose DDP resulted in the disappearance of large-sized SGs (Fig. 6D, F, and G). These results indicate that CD147 internalization and binds to G3BP1that might affect the assembly of SGs.

Fig. 6

Cisplatin-induced CD147 internalization formed a complex with G3BP1 accompanied by SGs depolymerization. A, B Laser confocal images showing the G3BP1-positive SG assembling upon treatment with cisplatin in Huh7 cells; DAPI-labeled nuclei. Scale bar: 20 μm. C Quantitation of SGs a quantity in A. Data is the means ± SD of three biological replicates. Count the number of SGs in ten images each time. Statistical significance by unpaired two-tailed Student’s t-test and two-way ANOVA (or mixed model); **, P < 0.01, *, P < 0.05. D Elyra 7 microscope images of HCCLM3 CtrlGFP and HCCLM3 △ECD-GFP cells transfected with mCherry-G3BP1. Scale bar: 10 μm. E Quantitation of speckles area percentage in D. Data are the means ± SD of three images in each group. The X-axis represents the area of the spot (0 μm2 ≤ area ≤ 1 μm2), and the Y-axis represents the percentage of the spot area. F Quantitation of speckles area percentage in D. Data are the means ± SD of three images in each group. The X-axis represents the area of the spot (1 μm2 < area ≤ 20 μm2), and the Y-axis represents the percentage of the spot area. G Elyra 7 microscope images of HCCLM3 △ECD-GFP cells transfected with mCherry-G3BP1. Scale bar: 10 μm

Combination administration enhances liver cancer treatment

To further demonstrate that overexpression of the CD147 intracellular segment could promote tumor chemotherapy resistance, we transfected Huh7 CtrlGFP and Huh7△ECD-GFP with luciferase virus and established a Huh7 xenograft model in non-obese diabetic (NOD) SCID gamma (NCG) mice. The fluorescence intensity of the tumors was observed after intraperitoneal injection of cisplatin on the fifth and 15th days (Fig. 7A). After the first injection of DDP, the intensity of fluorescence from tumors in Huh7 CtrlGFP was significantly reduced (P = 0.0028), but that of Huh7 △ECD-GFP was not significantly affected (Fig. 7B, C). Consistent with the above-mentioned results, the fluorescence intensity of tumors in the control group was reduced after the second injection of DDP (P = 0.02), whereas that in Huh7 △ECD-GFP remained unchanged (Fig. 7B, D). To further verify that cytoprotective autophagy leads to reduced sensitivity to chemotherapy, we established an HCCLM3 xenograft model in nude mice. The results showed that the tumor volume of HCCLM3 CtrlGFP mice treated with DDP alone (P = 0.0276) and dual administration groups (P = 0.0038; Fig. 7E, F) was significantly reduced compared to the saline group, while the tumor volume of HCCLM3 △ECD-GFP mice in DDP treatment alone was not significantly reduced. However, the tumor volume of HCCLM3 △ECD-GFP mice in the dual treatment group was significantly lower than that in the saline group (P = 0.0392) and the DDP administration alone group (P = 0.0302; Fig. 7E, F). HE and TUNEL assays showed tumor necrosis in HCCLM3 CtrlGFP mice treated with DDP alone and in combination (P = 0.0142), however, HCCLM3 △ECD-GFP mice treated with DDP alone had only slight tumor necrosis, while the necrotic area significantly increased in the dual administration groups (P = 0.0063; Figures S5A, B). Throughout the administration period, the weight of the mice steadily increased without any intergroup differences (Fig. 7G).

Fig. 7

Combination administration enhances liver cancer treatment. A Schematic diagram of periodic administration and monitoring of NCG mice. B Establishment of xenograft mouse models to evaluate the intensity of fluorescence using an in vivo imaging system. Fluorescence values ranged from 500 to 10,000 RLU. C, D Quantitation of fluorescence intensity after two times injection. Statistical significance by paired, two-tailed Student’s t-test; **, P < 0.01, n = 4 mice. E Establish a nude mouse subcutaneous transplant tumor model in vivo, and feed it with cisplatin or hydroxychloroquine for a total of 4 weeks. Measure the tumor nodule volume and mouse weight every 3 days to evaluate the anti-tumor effect of autophagy inhibition. F Quantitation of tumor size. Statistical significance by paired, two-tailed Student’s t-test; **, P < 0.01, *, P < 0.05, n = 5 mice. G Quantitation of mice body weight. Statistical significance by unpaired, two-tailed Student’s t test. H HE and CD44 staining of HCCLM3 CtrlGFP and △ECD-GFP tumors; Scale bar: 100 μm; 50 μm. I, J, K, L Quantitation of CD44 positive rate in HCCLM3 CtrlGFP and △ECD-GFP tumors treated with saline alone, DDP alone, and combined administration. Data are the means ± SD of four biological replicates in each group. Statistical significance by paired, two-tailed Student’s t-test; **, P < 0.01, *, P < 0.05. M CD147 C-terminal staining of clinical HCC tissues; Scale bar: 0.02 mm. N IF staining of clinical HCC tissues used by Akoya whole tissue slice multi-color fluorescence quantitative analyzer; Scale bar: 100 μm; 50 μm

CD44 is a widely distributed glycoprotein on the cell surface that can specifically bind to the extracellular matrix to affect the mutual adhesion between cells and the matrix, thereby affecting the differentiation of mesenchymal stem cells. As a vital surface marker of stem cells, CD44 is significantly up-regulated by autophagy [29]. Immunohistochemical (IHC) staining showed that the expression of CD44 in HCCLM3 △ECD-GFP was enhanced considerably compared to that in HCCLM3 CtrlGFP in saline- and cisplatin-treated groups (P = 0.0291; P = 0.0013; Fig. 7H-J). In the dual administration group using HCQ and DDP, the expression of CD44 in HCCLM3 △ECD-GFP was lower than that in saline- and cisplatin-treated groups, whereas there was no significant difference in every group of HCCLM3 CtrlGFP (P = 0.0432; P = 0.0165; Fig. 7K, L). Moreover, we also collected 12 clinical tissues of relapsed liver cancer with chemotherapy and adjacent lesions, along with 12 tissues of untreated liver cancer and adjacent lesions. The IHC results showed significant internalization of the CD147 intracellular segment in recurrent liver cancer tissues after chemotherapy (P = 0.0144; Fig. 7M, S5C-S5H). In previous studies, we also found that patients with a high positive rate of CD147 intracellular fragment expression had a significantly poor overall survival compared with patients with a low positive rate of CD147 intracellular fragment expression in 102 cases of HCC tissues [19]. High-resolution MS revealed that the abundance of CD147 intracellular peptide segments in liver cancer tissues that relapsed after chemotherapy was higher than that in adjacent lesions (Figures S5I, S5J). IHF results also showed significant co-localization of internalized CD147 intracellular segments with G3BP1 in the cytoplasm (Fig. 7N). These results indicate that the internalization of CD147 intracellular segments reduced the chemosensitivity of cancer cells to cisplatin and maintained their stemness in HCC, and combination administration could enhance liver cancer treatment via inhibited autophagy.