Methionine Metabolism Dictates PCSK9 Expression and Antitumor Potency of PD-1 Blockade in MSS Colorectal Cancer

Abstract

Nutrient metabolisms are vitally interrelated to cancer progression and immunotherapy. However, the mechanisms by which nutrient metabolisms interact to remodel immune surveillance within the tumor microenvironment remain largely unexplored. Here it is demonstrated that methionine restriction inhibits the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of cholesterol homeostasis and a potential target for cancer immunotherapy, in colorectal cancer (CRC) but not in the liver. Mechanistically, methionine is catabolized to S-adenosylmethionine (SAM), promoting mRNA transcription of PCSK9 through increased DNA methyltransferase 1 (DNMT1)-mediated DNA methylation and suppression of sirtuin 6 (SIRT6) expression. Furthermore, both PCSK9 inhibition and dietary methionine restriction (DMR) potentiate PD-1 blockade therapy and foster the infiltration of CD8⁺ T cells in Colon 26 tumor-bearing mice-a proficient mismatch repair (pMMR)/microsatellite stable (MSS) CRC model that exhibits limited response to anti-PD-1 therapy. Moreover, combining 5-fluorouracil (5-FU) chemotherapy with PCSK9 inhibition and PD-1 blockade further augments therapeutic efficacy for MSS CRC. The findings establish a mechanistic link between amino acid metabolism and cholesterol metabolism within the tumor microenvironment where tumor cells sense methionine to regulate PCSK9 expression, highlighting promising combination therapeutic strategies that may greatly benefit MSS CRC patients.

Keywords: MSS colorectal cancer; PCSK9; immunotherapy resistance; methionine metabolism.

Figure 1

Methionine deprivation inhibits PCSK9 expression in vivo. A) Immunoblotting analysis of PCSK9 in SW480 cells cultured with RPMI 1640 medium without individual amino acids for 6h. B) Densitometric quantification of the ratio of PCSK9 to β‐actin (n = 3). The ratio in complete medium group was set as 1. C–F) The expression and secretion of PCSK9 in both human and mouse CRC cells with methionine deprivation for 6 h and then methionine supplementation for 24h. Immunoblotting analysis of PCSK9 in human CRC cell lines (SW480, SW620, HCT116, and LoVo) C) and mouse CRC cell lines (MC38 and Colon 26) E). The densitometric quantification of the ratio of PCSK9 to β‐actin is calculated E). ELISA analysis of PCSK9 in culture supernatant of human CRC cell lines (SW480, SW620, HCT116, and LoVo) D) and mouse CRC cell lines (MC38 and Colon 26) F). G,H) Immunohistochemistry analysis of PCSK9 in tumors from MC38‐ (G, n = 9) and Colon 26‐ (H, n = 4 (ND), n = 8 (DMR)) bearing mice fed with normal diet and methionine restriction diet (left). The abundance of PCSK9 was assessed (right). I) Immunoblotting analysis of PCSK9 in tumors from Colon 26‐bearing mice fed with normal diet and methionine restriction diet. Densitometric quantification of the ratio of PCSK9 to β‐actin is calculated. J,K) Tumor volume of PCSK9‐KD (J, n = 10) or PCSK9‐KO (K, n = 9) MC38‐bearing mice fed with normal diet and methionine restriction diet. EAA, essential amino acid; NEEA, non‐essential amino acid; CM, complete medium; ND, normal diet; DMR, dietary methionine restriction. Data were analyzed by unpaired two‐tailed Student's t‐test B, D, F, G, and H) or two‐way ANOVA J and K). Error bars denote for the s.e.m. ns, not significant; **p < 0.01, ***p < 0.001.

Figure 2

Methionine promotes PCSK9 mRNA transcription. A,B) Relative mRNA level of PCSK9 in SW480 (A, n = 9) and SW620 (B, n = 9) cells with methionine deprivation for 6 h and then methionine supplementation (0, 50 µm) for 24h. C,D) Relative mRNA level of PCSK9 in SW480 (C, n = 9) and SW620 (D, n = 9) cells with methionine deprivation for 6 h and then methionine supplementation (0, 50 µm) together with actinomycin D (10 µg mL⁻¹) treatment for the indicated time were analyzed. The mRNA stability of PCSK9 was assessed. E) Immunoblotting analysis of PCSK9 in SW480 (upper panel) and SW620 (lower panel) cells with methionine deprivation for 6 h and then methionine supplementation (0, 50 µm) together with actinomycin D (10 µg mL⁻¹) treatment for 24h. F,G) Luciferase reporter assays of hPCSK9 promoter activity in SW480 (F, n = 3) and SW620 (G, n = 3) cells transfected with hPCSK9 promoter luciferase reporter plasmid together with Renilla luciferase plasmid, and then cultured with methionine deprivation for 6 h and supplementation (0, 50 µm) for 24h. The relative hPCSK9 promoter luciferase activity for methionine starvation group was set as 1. Data were analyzed by unpaired two‐tailed Student's t‐test A, B, F, and G) or two‐way ANOVA C and D). Error bars denote for the s.e.m. ns, not significant; ***p < 0.001.

Figure 3

Methionine is catabolized to SAM to promote PCSK9 expression. A) The mRNA level of MAT2A in tumor tissues (n = 476) and normal tissues (n = 41) of Colon Adenocarcinoma from TCGA database. B) Correlation between MAT2A and PCSK9 mRNA levels in tumor tissues (n = 476) of Colon Adenocarcinoma from the TCGA database was calculated using linear regression. C) Luciferase reporter assays of hPCSK9 promoter activity in SW480 cells transfected with hPCSK9 promoter luciferase reporter plasmid together with Renilla luciferase plasmid, and then cultured with methionine deprivation for 6 h and SAM addition (0, 500 µm) for 24 h (n = 3). The relative hPCSK9 promoter luciferase activity for methionine starvation group was set as 1. D) Relative mRNA level of PCSK9 in SW480 cells with methionine deprivation for 6 h and then SAM addition (0, 500 µm) for 24 h (n = 9). E,F) Immunoblotting analysis of PCSK9 in SW480 (upper panel) and SW620 (lower panel) cells with methionine deprivation for 6 h and then SAM E) or SAH F) supplementation for 24h. G,H) Immunoblotting analysis of PCSK9 in SW480 cells transfected shMAT2A plasmids and treated with methionine G) or SAM H) for 24h. I,J) Immunoblotting analysis of PCSK9 in SW480 (upper panel) and SW620 (lower panel) cells with methionine deprivation for 6 h, and then SAM I) or SAH J) supplementation together with AG‐270 (1 µm) treatment for 24h. K) Immunoblotting analysis of PCSK9 in SW480, HCT116, and LoVo cells with methionine deprivation for 6 h and then SAM addition (0, 100, 500 µm) together with actinomycin D (10 µg mL⁻¹) treatment for 24h. Data were analyzed by unpaired two‐tailed Student's t‐test (A, C, and D) or Pearson r (B). Error bars denote for the s.e.m. ***p < 0.001.

Figure 4

DNMT1‐mediated methylation promotes mRNA transcription of PCSK9. A,B) Luciferase reporter assays of hPCSK9 promoter activity in SW480 cells transfected with hPCSK9 promoter luciferase reporter plasmid together with Renilla luciferase plasmid, and then cultured with methionine deprivation for 6 h followed by methionine (A, n = 3) or SAM (B, n = 3) supplementation and 5‐azacytidine (5 µm) treatment for 24h. The relative hPCSK9 promoter luciferase activity for methionine starvation group was set as 1. C,D) Relative mRNA level of PCSK9 in SW480 cells with methionine deprivation for 6 h and then methionine (C, n = 9) or SAM (D, n = 6 or 9) supplementation together with 5‐azacytidine (5 µm) treatment for 24h. E,F) Immunoblotting analysis of PCSK9 in SW480 (upper panel) and SW620 (lower panel) cells with methionine deprivation for 6 h, and then methionine E) or SAM F) supplementation together with 5‐azacytidine (5 µm) treatment for 24h. G) The mRNA level of DNMT1 in tumor tissues (n = 476) and normal tissues (n = 41) of Colon Adenocarcinoma from TCGA database. H) Correlation between DNMT1 and PCSK9 mRNA levels in tumor tissues (n = 476) of Colon Adenocarcinoma from the TCGA database was calculated using linear regression. I) Luciferase reporter assays of hPCSK9 promoter activity in SW480 cells transfected with si‐DNMT1 (si‐NC served as a negative control) together with hPCSK9 promoter luciferase reporter plasmid and Renilla luciferase plasmid, and then cultured with methionine deprivation for 6 h followed by methionine or SAM supplementation for 24 h (n = 3). The relative hPCSK9 promoter luciferase activity for negative control was set as 1. J) Relative mRNA level of PCSK9 in SW480 cells transfected with si‐DNMT1 (si‐NC served as a negative control), and then cultured with methionine deprivation for 6 h and then methionine or SAM supplementation for 24 h (n = 9). K,L) Immunoblotting analysis of PCSK9 in SW480 (upper panel) and SW620 (lower panel) cells transfected with si‐DNMT1 (si‐NC served as a negative control), and then cultured with methionine deprivation for 6 h and methionine K) or SAM L) supplementation for 24h. Data were analyzed by unpaired two‐tailed Student's t‐test (A, B, C, D, G, I, and J) or Pearson r (H). Error bars denote for the s.e.m. ***p < 0.001.

Figure 5

Methionine inhibits SIRT6 through DNA methylation to promote PCSK9 mRNA transcription. A,b) Methylation level of SIRT6 A) and FOXO3 B) in SW480 cells with methionine deprivation for 6 h followed by methionine supplementation for 6h. C,D) Immunoblotting analysis of PCSK9, SIRT6, and FOXO3A in SW480 (C) and SW620 (D) cells with methionine deprivation for 6 h and methionine supplementation for 24h. E,F) Relative mRNA level of PCSK9 (E, n = 9) and SIRT6 (F, n = 9) in SW480 cells with methionine deprivation for 6 h and then methionine supplementation (0, 50 µm) for 24h. G) Relative mRNA level of SIRT6 in SW480 cells with methionine deprivation for 6 h and then methionine supplementation together with 5‐azacytidine (5 µm) treatment for 24 h (n = 9). H) Immunoblotting analysis of PCSK9 and Flag in SW480 transfected with Flag‐SIRT6 and then cultured with methionine deprivation for 6 h and methionine supplementation for 24h. I,J) Relative mRNA level of PCSK9 (I) and SIRT6 J) and in SW480 cells transfected with shSIRT6 and culture with methionine deprivation for 6 h and then methionine supplementation for 24h. K) Luciferase reporter assays of hPCSK9 promoter activity in SW480 cells transfected with shSIRT6 (shNC served as a negative control) together with hPCSK9 promoter luciferase reporter plasmid and Renilla luciferase plasmid, and then cultured with methionine deprivation for 6 h followed by methionine supplementation for 24 h (n = 3). The relative hPCSK9 promoter luciferase activity for negative control was set as 1. Data were analyzed by unpaired two‐tailed Student's t‐test (A, B, E, F, G, I, J, and K). Error bars denote for the s.e.m. **p < 0.01, ***p < 0.001.

Figure 6

Dietary methionine restriction potentiates PD‐1 blockade therapy for MSS CRC. A) Schematic representation of combined therapy of PD‐1 blockade with dietary methionine restriction to Colon 26 tumor‐bearing mice. B–D) Tumor volume B), tumor size C), and relative tumor weight D) of Colon 26‐bearing mice with anti‐PD‐1 therapy and dietary methionine restriction (n = 6). E) Immunohistochemistry analysis of CD8 in tumor tissues from Colon 26‐bearing mice with anti‐PD‐1 therapy and dietary methionine restriction. F) Number of CD8 T cells in tumor tissues from Colon 26‐bearing mice with anti‐PD‐1 therapy and dietary methionine restriction was calculated (n = 4 or 6). G) Flow cytometric analysis of intratumoral CD8⁺ cytotoxic T cells (TILs) in Colon 26‐bearing mice fed with a methionine restriction diet alone or in combination with anti‐PD‐1 therapy. H) Flow cytometric analysis of TNFα and Granzyme B in intratumoral CD8⁺T cells from Colon 26‐bearing mice fed with a methionine restriction diet alone or in combination with anti‐PD‐1 therapy. I) Schematic representation of CD8 T cell depletion treatment in Colon 26 tumor‐bearing mice. J) Tumor volume of Colon 26‐bearing mice with anti‐PD‐1 therapy, dietary methionine restriction, and CD8 T cell depletion (n = 7 or 8). Data were analyzed by two‐way ANOVA B and J) or unpaired two‐tailed Student's t‐test (D, F, G, and H). Error bars denote for the s.e.m. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

PCSK9 inhibition potentiates PD‐1 blockade therapy and 5‐FU chemotherapy for MSS CRC. A) Schematic representation of combined therapy of PD‐1 blockade with PCSK9 inhibition to Colon 26 tumor‐bearing mice. B) Tumor volume of Colon 26‐bearing mice with anti‐PD‐1 therapy and PCSK9 inhibition (n = 8 to 10). C) Immunohistochemistry analysis of CD8 in tumor tissues from Colon 26‐bearing mice with anti‐PD‐1 therapy and PCSK9 inhibition. D) Number of CD8 T cells in tumor tissues from Colon 26‐bearing mice with anti‐PD‐1 therapy and PCSK9 inhibition was calculated (n = 5 to 8). E) Schematic representation of combined therapy of 5‐FU chemotherapy with PD‐1 blockade and PCSK9 inhibition to Colon 26 tumor‐bearing mice. F–H) Tumor volume F), tumor size G), and relative tumor weight H) of Colon 26‐bearing mice with combined therapy of 5‐FU chemotherapy, PD‐1 blockade, and PCSK9 inhibition (n = 7). Data were analyzed by two‐way ANOVA (B and F) and unpaired two‐tailed Student's t‐test (D and H). Error bars denote for the s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001.