SETDB1 Methylates MCT1 Promoting Tumor Progression by Enhancing the Lactate Shuttle

Abstract

MCT1 is a critical protein found in monocarboxylate transporters that plays a significant role in regulating the lactate shuttle. However, the post-transcriptional modifications that regulate MCT1 are not clearly identified. In this study, it is reported that SETDB1 interacts with MCT1, leading to its stabilization. These findings reveal a novel post-translational modification of MCT1, in which SETDB1 methylation occurs at K473 in vitro and in vivo. This methylation inhibits the interaction between MCT1 and Tollip, which blocks Tollip-mediated autophagic degradation of MCT1. Furthermore, MCT1 K473 tri-methylation promotes tumor glycolysis and M2-like polarization of tumor-associated macrophages in colorectal cancer (CRC), which enhances the lactate shuttle. In clinical studies, MCT1 K473 tri-methylation is found to be upregulated and positively correlated with tumor progression and overall survival in CRC. This discovery suggests that SETDB1-mediated tri-methylation at K473 is a vital regulatory mechanism for lactate shuttle and tumor progression. Additionally, MCT1 K473 methylation may be a potential prognostic biomarker and promising therapeutic target for CRC.

Keywords: MCT1 methylation; SETDB1; lactate shuttle; tumor progression.

Figure 1

SETDB1 interacts with MCT1 and enhances its expression. a,b) Whole cell lysates (WCL) of SW480 and HT29 cells were collected for IP with anti‐MCT1 or anti‐SETDB1 antibody, followed by immunoblots (IB) analysis. c) The co‐localization of MCT1 and SETDB1 was identified by IF analysis using anti‐MCT1 and anti‐SETDB1 antibodies in SW480 cells. White scale bars, 5 µm. d) In vitro binding assay was performed. Purified Flag‐SETDB1 was incubated with His‐MCT1(444‐500aa) and pulled down using anti‐Flag beads, followed by IB analysis. e–h) Protein and mRNA expression of MCT1 were detected by IB assays and qRT‐PCR assays in SW480 and HT29 cells silenced with control (shNC) or SETDB1 shRNA (#1 and #2). i,j) Protein and mRNA expression of MCT1 were measured by IB assays and qRT‐PCR assays in SW480 cells transfected with Vector, Flag‐SETDB1 (WT), and Flag‐SETDB1 (H1224K) plasmids. k,l) Protein and mRNA expression of MCT1 were determined by IB assays and qRT‐PCR assays in SW480 cells treated with Mithramycin A at the indicated concentrations. m) Colorectal tumors and paired normal tissues were extracted and subjected to detection of MCT1 and SETDB1 protein expression by IB analysis. All immunoblots are performed three times, independently, with similar results. f,h,j,l) Data are represented as mean ± s.d. ns means no significant, by one‐way analysis of variance (ANOVA) with Tukey's test.

Figure 2

SETDB1 represses the autophagic degradation of MCT1. a) The degradation of MCT1 was detected in shSETDB1NC and shSETDB1#1 SW480 cells treated with CHX (100 µg mL⁻¹) for 0, 4, 8, or 12 h by CHX‐chase assay. b) Quantification of the relative protein level of MCT1 in (a). c) The protein level of MCT1 was examined in SW480 cells in the presence of different inhibitors MG132 (10 × 10⁻⁶ m), CQ (50 × 10⁻⁶ m), or 3‐MA (5 × 10⁻³ m). d) The degradation of MCT1 was evaluated by CHX‐chase assay in ATG5 WT or ATG5 KO SW480 cells. e) Quantification of the relative protein level of MCT1 in (d). f) The degradation of MCT1 was evaluated by CHX‐chase assay in Beclin 1 WT or Beclin 1 KO SW480 cells. g) Quantification of the relative protein level of MCT1 in (f). h,i) The protein level of MCT1 in WT and ATG5 or Beclin 1 KO SW480 cells treated with dimethyl sulfoxide or Mithramycin A (100 × 10^{− 9} m, 24 h). All immunoblots were performed three times, independently, with similar results. b,e,g) Data are represented as mean ± s.d. **p < 0.01, ****p < 0.0001, by two‐way ANOVA with Tukey's test.

Figure 3

SETDB1 induces tri‐methylation of lysine 473 on MCT1. a) WCL collected from SW480 and HT29 cells silenced with control (ShNC) or SETDB1 ShRNA (#1, #2) were subjected to IP assay with anti‐MCT1 antibody, followed by IB analysis. b) WCL collected from SW480 and HT29 cells transfected with Vector, Flag‐SETDB1 (WT), and Flag‐SETDB1 (H1224K) plasmids were subjected to IP assay with anti‐MCT1 antibody, followed by IB analysis. c) HEK293T cells transfected with HA‐MCT1 WT or mutant plasmids as indicated, then transfected with Vector or Flag‐SETDB1, WCL were collected for IP with anti‐HA beads, followed by IB analysis. d) Secondary mass spectrometry result of lysine 473 methylation residue. e) SW480 ^{MCT1 KO} cells transfected with HA‐MCT1 WT or K473R plasmid as indicated, then transfected with Vector or Flag‐SETDB1, WCL were collected for IP with anti‐HA beads, followed by IB analysis. f) Different peptides were added into PVDF membranes at indicated concentrations, followed by a dot blot assay using MCT1 K473 specific tri‐methylation antibody. g) In vitro methylation assay was performed. Purified Flag‐SETDB1 was incubated with His‐MCT1(444‐500aa) in the presence of S‐adenosyl‐L‐methionine, followed by IB analysis to analyze MCT1 methylation using MCT1 K473‐specific tri‐methylation antibody. h) WCL collected from SW480 and HT29 cells silenced with control (ShNC) or SETDB1 ShRNA (#1, #2) were subjected to IB assay with MCT1 K473‐specific tri‐methylation antibody. All immunoblots were performed three times, independently, with similar results.

Figure 4

MCT1 K473 tri‐methylation blocks Tollip‐mediated autophagic degradation of MCT1. a) The degradation of MCT1 was detected in SW480 ^{MCT1 KO} cells stably expressing MCT1 WT or MCT1 K473R cells treated with CHX (100 µg mL⁻¹) for 0, 4, 8, or 12 h by CHX‐chase assay. b) Quantification of relative protein level of MCT1 in (a). c) HEK293T cells were transfected with HA‐MCT1 and indicated Flag‐tagged cargo receptors, WCL were collected for IP with anti‐Flag beads, followed by IB analysis. d) The degradation of MCT1 was evaluated by CHX‐chase assay in Tollip WT or Tollip KO SW480 cells. e) Quantification of the relative protein level of MCT1 in (d). f) WCL collected from SETDB1 knockdown SW480 and HT29 cells were subjected to IP assay with anti‐MCT1 antibody, followed by IB analysis. g) WCL collected from SW480 cells transfected with Vector, Flag‐SETDB1 (WT), and Flag‐SETDB1 (H1224K) plasmids were subjected to IP assay with anti‐MCT1 antibody, followed by IB analysis. h) HEK293T cells were co‐transfected with Flag‐Tollip and Vector, HA‐MCT1 WT or HA‐MCT1 K473R, and WCL was collected for IP with anti‐HA beads, followed by IB analysis. i) The protein level of MCT1 in Tollip WT and Tollip KO SW480 cells treated with DMSO or Mithramycin A (100 × 10⁻⁹ m, 24 h). All immunoblots were performed three times, independently, with similar results. a,b) Data are represented as mean ± s.d. ****p < 0.0001, by two‐way ANOVA with Tukey's test.

Figure 5

MCT1 K473 tri‐methylation promotes tumor glycolysis and M2‐like polarization of TAMs by regulating lactate transport. a) Lactate production was measured in the conditioned medium (CM) of SW480 ^{MCT1 KO} cells stably expressing Vector, MCT1 WT, or MCT1 K473R, n = 3. b) Glucose uptake was measured in SW480 ^{MCT1 KO} cells stably expressing Vector, MCT1 WT, or MCT1 K473R, n = 3. c,d) Bioenergetic analysis was performed with the Seahorse XF24 analyzer platform. ECAR of SW480 ^{MCT1 KO} cells stably expressing Vector, MCT1 WT, or MCT1 K473R was measured and calculated, n = 3. e) WCL collected from SW480 ^{MCT1 KO} cells stably expressing Vector, MCT1 WT, or MCT1 K473R, followed by IB assay. f) The relative mRNA levels of VEGF and ARG1 macrophage markers in BMDMs treated with indicated CM, n = 3. g) The relative mRNA levels of VEGF and ARG1 macrophage markers in BMDMs treated with indicated CM containing with or without lactate (5 × 10⁻³ m), n = 3. h) WCL collected from BMDMs treated by the indicated CM, followed by IB assay. i) A syngeneic tumor model was performed by injecting MCT1‐3′UTR CT26 cells stably expressing Vector, mus‐MCT1 WT, or mus‐MCT1 K467R cells into BALB/c mice. n = 5 mice. j,k) Quantification of tumor weight and volume of tumors generated in (i). l,m) Flow cytometry analysis of macrophage polarization of tumors generated in (i). n) Representative images for infiltration of intratumor CD206⁺ and F4/80⁺ cells by IF. White scale bars, 20 µm. All immunoblots were performed three times, independently, with similar results. Data are represented as mean ± s.d. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns means no significant, by a,b,j,m) one‐way ANOVA with Tukey's test and d,f,g,k) two‐way ANOVA with Tukey's test.

Figure 6

MCT1 K473 tri‐methylation is required for SETDB1‐mediated tumor glycolysis and M2‐like polarization of TAMs. a) Lactate production was measured in the conditioned medium (CM) of SW480 ^{MCT1 KO} cells stably expressing MCT1 WT or MCT1 K473R and transfected with Vector or Flag‐SETDB1, n = 3. b) Glucose uptake was measured in SW480 ^{MCT1 KO} cells stably expressing MCT1 WT or MCT1 K473R and transfected with Vector or Flag‐SETDB1, n = 3. c,d) Bioenergetic analysis was performed with the Seahorse XF24 analyzer platform. ECAR of SW480 ^{MCT1 KO} cells stably expressing MCT1 WT or MCT1 K473R and transfected with Vector or Flag‐SETDB1 was measured and calculated, n = 3. e) WCL collected from SW480 ^{MCT1 KO} cells stably expressing MCT1 WT or MCT1 K473R and transfected with Vector or Flag‐SETDB1, followed by IB assay. f) The relative mRNA levels of VEGF and ARG1 macrophage markers in BMDMs were treated with indicated CM, n = 3. g) A syngeneic tumor model was performed by injecting MCT1‐3′UTR CT26 cells stably expressing MCT1 WT or MCT1 K473R cells and overexpression Vector or SETDB1 into BALB/c mice. n = 5 mice. h,i) Quantification of tumor weight and volume of tumors generated in (g). j,k) Flow cytometry analysis of macrophage polarization of tumors generated in (g). All immunoblots were performed three times, independently, with similar results. Data are represented as mean ± s.d. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns means no significant, by a,b,h,k) one‐way ANOVA with Tukey's test and d,f,i) two‐way ANOVA with Tukey's test.

Figure 7

MCT1 K473 tri‐methylation is positively related to CRC and has prognostic significance in CRC patients. a) MCT1 K473 tri‐methylation IHC staining score was detected in tumor and adjacent tissues, n = 79. Student's two‐tailed t‐test, p < 0.001. b) Representative image of IHC staining for SETDB1, MCT1 K473 tri‐methylation, and CD206 in colorectal cancer. Black scale bar, 50 µm . c) Scatter plot of the IHC staining scores for SETDB1, MCT1 K473 tri‐methylation, and CD206 in CRC, n = 80. All p and r values were calculated with Spearman's r test. c) Quantitative IHC staining score showing the correlation of SETDB1 and MCT1 K473 tri‐methylation. Chi‐square test, p < 0.0001. d) Correlation between SETDB1 and MCT1 K473 tri‐methylation expression was determined by Pearson correlation coefficient test, p < 0.0001. e) Kaplan–Meier analysis of overall survival in a set of 80 colorectal cancer patients according to MCT1 K473 tri‐methylation expression. Log‐rank test, p = 0.0310. f,g) Quantitative IHC staining score showing the correlation between MCT1 K473 tri‐methylation and TNM stage or N stage using microarray of colorectal cancer specimen. Chi‐square test, p = 0.0155 and p = 0.0352, respectively. h) The working model of SETDB1‐mediated MCT1 K473 tri‐methylation.