Lactate Activates the HCAR1/β-Arrestin2/PP2A Signaling Axis to Mediate STAT1/2 Dephosphorylation and Drive Osteosarcoma Progression

Abstract

The "Warburg effect", a hallmark of Osteosarcoma(OS), results in lactate accumulation due to aerobic glycolysis. The role and underlying mechanisms of lactate in OS are not well understood. Herein, the lactate-activated hydroxycarboxylate receptor 1(HCAR1) is found to promote OS progression via inhibiting the transcription of anti-oncogene downstream of STAT1/2. The phosphorylation level of STAT1/2 holds considerable significance for transcriptional activity. In this study, protein phosphatase 2A(PP2A) is identified as the tyrosine phosphatase of STAT1/2. Lactate-activated HCAR1, facilitating PP2A interaction with phosphorylated STAT1/2 via β-Arrestin 2, resulting in STAT1/2 dephosphorylation, a key process linked to the aggressive behavior of OS. Using PP2A inhibitor Endothall can abolish the dephosphorylation effect of HCAR1 on STAT1/2, inhibit cancer cell proliferation, migration, and cell cycle, and promote apoptosis. Moreover, the combination of Endothall and Cisplatin is high synergistic in treating OS. In conclusion, the study elucidates the pro-oncogenic role of lactate-activated HCAR1 in OS.

Keywords: HCAR1; STAT1/2; lactate; osteosarcoma; β‐Arrestin 2.

Figure 1

HCAR1 is significantly upregulated in OS and inversely correlated with poor prognosis. A–C) HCAR1 mRNA and protein levels in OS and other sarcoma tissues (including Chondrosarcoma, Ewing sarcoma, and Liposarcoma) by qRT‐PCR and Western blot. D–F) HCAR1 mRNA and protein levels in OS and adjacent tissues across 65 samples by qRT‐PCR and Western blot. G) Five‐micrometer (5‐µm) sections analyzed by IHC using anti‐HCAR1 antibodies. The quantitative analyses (Negative and + = low expression, + + and + + + = high expression) were performed for each sample (also see Figure S2, Supporting Information). H) Kaplan–Meier curve showing the correlation between HCAR1 expression levels and recurrence‐free survival rate (RFS) in OS patients. I) Kaplan–Meier curve showing the correlation between HCAR1 expression levels and overall survival (OS) in OS patients. Statistical analysis was performed using one‐way ANOVA (A,C) and unpaired t‐test (D,F). Error bars show means ± SD. *p < 0.05, **p < 0.01,***P < 0.001, and ****P < 0.0001. Scale bars, 200 or 50 µm.

Figure 2

Lactate activating HCAR1 drives OS cell proliferation, migration, and invasion. A–C) HCAR1 mRNA and protein levels in BMSCs and OS cell lines by qRT‐PCR and Western blot. D–G) Validation of the mRNA and protein expression of HCAR1 gain and loss in MG63 and 143B cells by qRT‐PCR and western blotting assays (also see Figure S3F,G, Supporting Information). H,I) CCK8 assay and proliferation activity of the indicated OS cells featuring over‐expression or knockdown HCAR1. J–L) Soft agar clone formation assay as well as the size and number of the tumor sphere‐formation of the indicated OS cells, which over‐expression or knockdown HCAR1 (also see Figure S3H,I, Supporting Information). M–O) Transwell assays to evaluate the effects of HCAR1 on the migration and invasion of the indicated OS cells. Representative images and quantification of relative migrated cells are presented. P–R) Cell cycle was evaluated by the effects of HCAR1 function using flow cytometry in the indicated OS cells. Evaluated HCAR1 significantly promoted cell population at G2/M phase. Statistical analysis was performed using one‐way ANOVA (A,C–E,H,I,K,L,N,O,Q,R). Error bars show means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and****P < 0.0001.

Figure 3

Lactate activating HCAR1 suppresses STAT1/2 transcriptional activity, thereby downregulating the expression of antioncogene downstream of STAT1/2. A) CRISPR‐Cas 9 performed to construct HCAR1‐knockout 143B cells. B) Validation of the knockout efficiency of HCAR1 in 143B cells by Western blot. C,D) RNA sequencing performed in wild‐type cells and HCAR1 knockout cells after lactate stimulation. Difference analysis of the transcriptome sequencing data was performed using the R Statistical Software. E) KEGG enrichment performed on the differential genes. F) ChIP‐X Enrichment Analysis 3 (ChEA 3) enriching the TFs for differential genes. G) Cytoscape visualizing co‐regulatory network of TFs. H) Differential analysis of the transcriptome sequencing data of STAT1/2 target genes. I) Validation of the effect of HCAR1 gain and loss on the mRNA levels of STAT1/2 and its target genes by qRT‐PCR. Statistical analysis was performed using one‐way ANOVA. (I). Error bars show means ± SD. ***P < 0.001, and ****P < 0.0001; NS, not significant.

Figure 4

In OS cells, β‐Arrestin2 mediates the phosphorylation inhibition of STAT1/2 by lactate/HCAR1. A,B) The HCAR1/G_i/o signaling pathway of MG63 (A) and 143B (B) cells blocked by PT, and the phosphorylation level of STAT1/2 detected by Western blot. C,D) CRISPR‐Cas9 performed to knock out the 13 base pairs in β‐Arrestin1 and the 5 base pairs in β‐Arrestin2, and DNA sequencing detecting the knockout effect of β‐Arrestin1 (C) or β‐Arrestin2 (D) in 143B cells. E,F) Validation of the knockout effect of β‐Arrestin1(E) and β‐Arrestin2 (F) in 143B cells by Western blot. G,H) Western blot performed to evaluate the effect of β‐Arrestin1 knockout (G) or β‐Arrestin2 knockout (H) on the Phosphorylation levels of STAT1/2. Statistical analysis was performed using two‐way ANOVA test, followed by post hoc testing with Tukey's Honestly Significant Difference (HSD) test (A,B,G,H). Error bars show means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; NS, not significant.

Figure 5

Lactate activating HCAR1 enhances PP2Aα binding to STAT1/2 via β‐Arrestin2. A,B) Immunoprecipitation experiments performed to assess the effect of lactate/ HCAR1 on the endogenous interaction of STAT1/2 and PP2Aα, β‐Arrestin1, β‐Arrestin2, PP2Aα, and TC45 of the indicated OS cells. 143B cell lysates were subjected to immunoprecipitation with anti‐STAT1/2, anti‐β‐Arrestin1, anti‐β‐Arrestin2, anti‐PP2Aα or anti‐TC45 antibody. The immunoprecipitates were then detected using the indicated antibodies. C–F) Immunoprecipitation experiments were performed to assess the effect of β‐Arrestin1 and β‐Arrestin2 on the endogenous interaction of STAT1/2 and PP2Aα, TC45 of the indicated OS cells. 143B cell lysates were subjected to immunoprecipitation with control IgG, anti‐STAT1/2, anti‐PP2Aα, or anti‐TC45 antibody. The immunoprecipitates were then detected using the indicated antibodies. G,H) Detection of the effect of lactate/HCAR1 on the relationship between STAT1/2 and PP2Aα in indicated OS cells by in situ PLA. PLA signal (Red fluorescence) appeared in situ of 143B cell. Statistical analysis was performed using two‐way ANOVA, followed by post hoc testing with Tukey's Honestly Significant Difference (HSD) test (A,B). Error bars show means ± SD. **P < 0.01, ****P < 0.0001; NS, not significant. Scale bars, 10 µm.

Figure 6

Inhibiting the process of PP2A dephosphorylating STAT1/2 could block the pro‐cancer effects of Lactate/HCAR1/β‐Arrestin2. A,B) MG63 and 143B cells treated with 10 mm lactate and 20 µM Endothall for 24 h, and the phosphorylation levels of STAT1/2 in the indicated OS cells measured by Western blot. C–E) Soft agar clone formation assay performed, as well as the size and number of the tumor sphere‐formation of the indicated OS cells treated with 10 mm lactate or 20 µm Endothall. F–H) Transwell assays performed to evaluate the effects of Endothall on the migration and invasion of the indicated OS cells. Representative images and quantification of relative migrated cells are presented. I,J) Cell cycle and apoptosis evaluated by the effects of Endothall intervention using flow cytometry in the indicated OS cells. Endothall significantly decreased the cell population at G2/M phase and increased the proportion of apoptotic cells. Statistical analysis was performed using two‐way ANOVA, followed by post hoc testing with Tukey's Honestly Significant Difference (HSD) test (A,B,D,E,G,H,I,J). Error bars show means ± SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001; NS, not significant. Scale bars, 100 or 200 µm.

Figure 7

HCAR1 knockout or PP2A inhibitor promotes OS cell apoptosis in vitro and suppresses OS cell proliferation and metastasis in vivo. A,B) Using cisplatin as a reference (positive drug), the killing efficacy of HCAR1 knockout or Endothall on 143B cells evaluated by flow cytometry. C) Eight‐week‐old female BALB/c nude mice subjected to subcutaneous implantation into the dorsolateral side of the flank region for the indicated cell, and treated with Endothall, cisplatin, or a combination of both for two weeks on day 14. Representative images of the primary tumors from each group for twenty‐eight days are presented. D,E) Primary tumor volume measured every three days. Primary tumors were removed and weighed in each group. F) HCAR1 knockout 143B cells injected into nude mice through the tail vein, and the treatment of Endothall or cisplatin, or a combination of both performed on day 7. Bioluminescent imaging was performed on day 21. G,H) Bioluminescent imaging performed and total fluorescence computed to evaluate tumor growth and metastasis in the indicated tumor‐bearing mice. Statistical analysis was performed using multifactor ANOVA, followed by post hoc testing with Tukey's Honestly Significant Difference (HSD) test (B,D,E,H). Error bars show means ± SD. *P < 0.05, ****P < 0.0001; NS, not significant.

Figure 8

In OS clinical samples, HCAR1 is co‐expressed with Ki67 but separated with phosphorylated STAT1/2. The expression of HCAR1, Ki67, and phosphorylated STAT1/2 in OS clinical detected by immunohistochemistry. Scale bars, 40 or 200 µm. The function and mechanism of lactate‐activated HCAR1 mediating STAT1/2 dephosphorylation to promote OS development.