Modulation of EZH2 Expression by MEK-ERK or PI3K-AKT Signaling in Lung Cancer Is Dictated by Different KRAS Oncogene Mutations

Abstract

EZH2 overexpression promotes cancer by increasing histone methylation to silence tumor suppressor genes, but how EZH2 levels become elevated in cancer is not understood. In this study, we investigated the mechanisms by which EZH2 expression is regulated in non-small cell lung carcinoma cells by oncogenic KRAS. In cells harboring KRAS(G12C) and KRAS(G12D) mutations, EZH2 expression was modulated by MEK-ERK and PI3K/AKT signaling, respectively. Accordingly, MEK-ERK depletion decreased EZH2 expression in cells harboring the KRAS(G12C) mutation, whereas PI3K/AKT depletion decreased EZH2 expression, EZH2 phosphorylation, and STAT3 activity in KRAS(G12D)-mutant cell lines. Combined inhibition of EZH2 and MEK-ERK or PI3K/AKT increased the sensitivity of cells with specific KRAS mutations to MEK-ERK and PI3K/AKT-targeted therapies. Our work defines EZH2 as a downstream effector of KRAS signaling and offers a rationale for combining EZH2 inhibitory strategies with MEK-ERK- or PI3K/AKT-targeted therapies to treat lung cancer patients, as stratified into distinct treatment groups based on specific KRAS mutations.

Figure 1

EZH2 was higher in tumors with a KRAS^G12C mutation compared with other KRAS mutant, and KRAS knockdown downregulates EZH2 expression in cell lines with KRAS mutant. A, Box plot of EZH2 expression of lung adenocarcinoma tumors with KRAS mutant (**P < 0.004). B, Representative photomicrographs of EZH2 immunohistochemical expression in NSCLC with different amino acids substituted on the KRAS mutant. C, EZH2 expression in NSCLC (left) and HBEC cell lines (right). D, KRAS and EZH2 expression in NSCLC cell lines upon knockdown of KRAS by treatment with siKRAS.

Figure 2

Disruption of the signaling MEK-ERK pathway affects EZH2 expression in NSCLC cell lines with mutant KRAS^G12C, and the combination of MEK1i with EZH2i results in a significantly increased sensitivity to MEK-ERK targeted therapy in cells expressing mutant KRAS^G12C and KRAS^G12S. A and B, EZH2, MAPK P44/42, and phospho-MAPK P44/42 expression in NSCLC and HBEC cell lines. KRAS^WT and KRAS mutants were treated with different doses of the MEK1i AZD6244 (0, 0.5, and 1.0 μM). C, EZH2 and MEK1/2 expression in NSCLC cell lines upon knockdown of MEK1 expression by treatment with siMEK1. D, EZH2 inhibition with DZNep in combination with MEK1i. (Data are graphed as the mean percent increase ± percent standard deviation). Treatment with DZNep decrease AZD6244 IC_50, 7.2-fold (P < 0.03) in HCC44 cells, a 3.6-fold (P < 0.05) in H23 cells, and a 2.6-fold (P < 0.05) in A549 cells. E, Athymic nude mice were inoculated with H23 cell lines expressing KRAS^G12C mutant and then treated with vehicle, DZNep, AZD6244, or a combination of DZNep plus AZD6244. Tumor volume was determined for each treatment. (*P < 0.05; **P < 0.03, ***P < 0.001).

Figure 3

Disruption of the PI3K/AKT pathway affects EZH2 expression in NSCLC cell lines with mutant KRAS^G12D and KRAS^G12S, and the combination of AKTi with EZH2i results in a significant increased sensitivity in vitro and in vivo to PI3K/AKT targeted therapy in cells expressing mutant KRAS^G12D. A and B, EZH2, AKT, and phospho-AKT expression in NSCLC and HBEC cell lines. KRAS^WT and KRAS mutants were treated with different doses of the AKTi MK2206 (0, 25, and 50 nM). C, EZH2 and AKT expression in NSCLC cell lines upon knockdown of AKT expression by treatment with siAKT. D, Pharmacologic inhibition of EZH2 with DZNep in combination with AKT inhibition. (Data are graphed as the mean percent increase ± percent standard deviation). Treatment with DZNep decrease MK2206 IC₅₀, 2.0-fold (P < 0.05) in cells expressing KRAS^G12D and a 2.2-fold (P < 0.05) in cells expressing KRAS^G12S. E, Athymic nude mice were inoculated with HCC461 cell lines expressing KRAS^G12D and then treated with vehicle, DZNep, MK2206, or a combination of DZNep plus MK2206. Tumor volume was determined for each treatment. (*P < 0.05; **P < 0.03, ***P < 0.001).

Figure 4

EZH2 expression is regulated by MEK-ERK and PI3K/AKT signaling pathways in colon and pancreatic tumor KRAS mutants. A, top panel, EZH2, MAPK P44/42, and phospho-MAPK P44/42 expression in colon and pancreatic cell lines. KRAS^WT and KRAS mutants were treated with different doses of the MEK1i AZD6244 (0, 0.5, and 1.0 μM). A, bottom, EZH2, AKT, and phospho-AKT expression in colon and pancreatic cell lines. KRAS^WT and KRAS mutants were treated with different doses of the AKTi MK2206 (0, 25, and 50 nM). B, top, EZH2 inhibition with DZNep in combination with MEK1i. (Data are graphed as the mean percent increase ± percent standard deviation). Treatment with DZNep caused a 16-fold (P < 0.03) decrease in the AZD6244 IC₅₀ in MIA PaCa2 cells. B, bottom, EZH2 inhibition with DZNep in combination with AKTi. (Data are graphed as the mean percent increase ± percent standard deviation). Treatment with DZNep caused a decrease in MK2206 IC₅₀, 10.6-fold (P < 0.03) in cells expressing KRAS^G12C, a 6.4-fold (P < 0.05) in cells expressing KRAS^G12D and a 6.8-fold (P < 0.05) in cells expressing KRAS^G12D.

Figure 5

AKT depletion decreased pS21-EZH2, phospho-STAT3, and methyl K levels strongly in cell lines expressing KRAS^G12D mutants. A and B, EZH2, pS21-EZH2, STAT3, phospho-STAT3, and methyl K level expression in NSCLC cell lines and HBEC cell lines. KRAS^WT and KRAS mutants were treated with different doses of the AKTi MK2206 (0, 25, and 50 nM). C, EZH2, pS21-EZH2, STAT3, phospho-STAT3, and methyl K level expression in NSCLC cell lines expressing KRAS^G12C and KRAS^G12D mutants upon knockdown of AKT1, AKT2, and AKT3 expression by treatment with siAKT. D, EZH2, pS21-EZH2, STAT3, phospho-STAT3, and methyl K level expression in NSCLC cell lines expressing KRAS^G12C and KRAS^G12D mutants upon knockdown of EZH2. E, Proposed model showing EZH2 as a downstream effector of KRAS signaling in malignant cell KRAS mutants.