Histone methyltransferase SETD1A interacts with HIF1α to enhance glycolysis and promote cancer progression in gastric cancer

Abstract

Growing tumors alter their metabolic profiles to support the increased cell proliferation. SETD1A, a histone lysine methyltransferase which specifically methylates H3K4, plays important roles in both normal cell and cancer cell functions. However, the function of SETD1A in gastric cancer (GC) progression and its role in GC metabolic reprogramming are still largely unknown. In the current study, we discovered that the expression of SETD1A was higher in GC tumor specimens compared to surrounding nontumor tissues. Upregulation of SETD1A increased GC cell proliferation, whereas downregulation of SETD1A inhibited GC cell proliferation. Furthermore, knockdown of SETD1A reduced glucose uptake and production of lactate and suppressed glycolysis by decreasing the expression of glycolytic genes, including GLUT1, HK2, PFK2, PKM2, LDHA, and MCT4. Mechanistically, SETD1A interacted with HIF1α to strengthen its transactivation, indicating that SETD1A promotes glycolysis through coactivation of HIF1α. SETD1A and HIF1α were recruited to the promoter of HK2 and PFK2, where SETD1A could methylate H3K4. However, knockdown of SETD1A decreased the methylation of H3K4 on HK2 and PFK2 promoter and reduced HIF1α recruitment necessary to promote transcription of glycolytic genes. Inhibition of HIF1α decelerated SETD1A-enhanced GC cell growth. In additional, there was a linear correlation between SETD1A and several key glycolytic genes in human GC specimens obtained from TCGA dataset. Thus, our results demonstrated that SETD1A interacted with HIF1α to promote glycolysis and accelerate GC progression, implicating that SETD1A may be a potential molecular target for GC treatment.

Keywords: HIF1α; gastric cancer; glycolysis; histone methyltransferase SETD1A; progression.

Fig. 1

SETD1A was overexpressed in human GC specimens and predicated poor outcome. (A) Analysis of the protein of SETD1A in 18 pairs of GC specimens and surrounding nontumor tissues. (B) Analysis of the expression of SETD1A in normal (n = 36) and GC (n = 408) specimens from TCGA dataset obtained by GEPIA. (C) Analysis of the overall survival rate in SETD1A‐high and SETD1A‐low expression GC patients from GEO dataset. *P < 0.05.

Fig. 2

SETD1A promotes gastric cancer cell proliferation. (A, B) Overexpression of SETD1A increased BGC‐823 and AGS cell proliferation. BGC‐823 and AGS cells were transfected with SETD1A plasmid for 48 h and then seeded into 96‐well plates for CCK‐8 assays (mean ± SEM; n = 4; Student’s t‐test). (C, D) Knockdown of SETD1A decreased BGC‐823 and AGS cell proliferation. Cells were transfected with SETD1A siRNAs for 48 h and then seeded into 96‐well plates for CCK‐8 assays (mean ± SEM; n = 4; Student’s t‐test). (E, F) Stable SETD1A‐knockdown BGC‐823 and AGS cells grown slowly compared to control cells (mean ± SEM; n = 4; Student’s t‐test). (mean ± SEM; n = 4; Student’s t‐test) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3

Downregulation of SETD1A decreases glycolysis in BGC‐823 cells. (A–C) Glucose uptake (A), lactate production (B), and pH values (C) of SETD1A‐knockdown cells were reduced under normoxic (N) and hypoxic (H) conditions for 24 h compared to control BGC‐823 cells (mean ± SEM; n = 3; Student’s t‐test). (D) The protein levels of HK2 and LDHA were remarkedly reduced in SETD1A‐knockdown BGC‐823 cells under normoxic (N) and hypoxic (H) conditions for 24 h. (E–J) The mRNA levels of GLUT1 (E), HK2 (F), PFK2 (G), PKM2 (H), LDHA (I), and MCT4 (J) were remarkedly reduced in SETD1A‐knockdown BGC‐823 cells under normoxic (N) and hypoxic (H) conditions for 24 h (mean ± SEM; n = 4; Student’s t‐test). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4

SETD1A cooperates with HIF1α to enhance HIF1α transactivation. (A) SETD1A cooperated with HIF1α to enhance the HK2 and PFK2 promoter–reporter activity (mean ± SEM; n = 3; Student’s t‐test). (B) Downregulation of SETD1A decreased the HK2 and PFK2 promoter activity in BGC‐823 cells. SETD1A‐knockdown and control BGC‐823 cells were transfected with HRE‐Luc under normoxic (N) and hypoxic (H) conditions. Luciferase activity was measured 48 h after transfection (mean ± SEM; n = 3; Student’s t‐test). (C) Co‐IP analysis of the interaction between Flag‐SETD1A and HA‐HIF1α. (D) Co‐IP analysis of the interaction between endogenous SETD1A and HIF1α in BGC‐823 cells. (E) Downregulation of SETD1A did not change the expression of HIF1α and global H3K4me3 protein in BGC‐823 cells. (F, G) Downregulation of SETD1A reduced the levels of HIF1α (F), SETD1A (G), and H3K4me3 (H) on the HK2 and PFK2 promoter in BGC‐823 cells. ChIP analysis of the levels of HIF1α (F), SETD1A (G), and H3K4me3 (H) on the HK2 and PFK2 promoter in SETD1A‐knockdown and control BGC‐823 cells under normoxic (N) or hypoxic (H) condition (mean ± SEM; n = 3; Student’s t‐test). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5

Inhibition of HIF1α suppresses SETD1A‐enhanced GC cell proliferation. (A, B) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced BGC‐823 cell proliferation (mean ± SEM; n = 3; Student’s t‐test). (C, D) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced AGS cell proliferation (mean ± SEM; n = 4; Student’s t‐test). **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6

Knockdown of SETD1A reduces gastric cancer cell tumorigenesis. (A, B) Downregulation of SETD1A in BGC‐823 cells inhibited xenograft tumor growth (A) in nude mice and reduced tumor weight (B) (mean ± SEM; n = 6; Student’s t‐test). (C) The mRNA levels of the GLUT1, HK2, PFK2, PKM2, LDHA, and MCT4 were reduced in SETD1A‐knockdown tumors (mean ± SEM; n = 6; Student’s t‐test). *P < 0.05, **P < 0.01.

Fig. 7

The expression of SETD1A is positively correlated with the expression of glycolytic genes in human gastric cancer specimens from TCGA dataset. Linear regression of SETD1A and glycolytic genes GLUT1 (A), HK2 (B), PFK2 (C), PKM (D), and MCT4 (E) using GC samples from TCGA database obtained by GEPIA.