Nrf2 Activation Sensitizes K-Ras Mutant Pancreatic Cancer Cells to Glutaminase Inhibition

Abstract

Pancreatic cancer remains intractable owing to the lack of effective therapy for unresectable cases. Activating mutations of K-ras are frequently found in pancreatic cancers, but these have not yet been targeted by cancer therapies. The Keap1-Nrf2 system plays a crucial role in mediating the oxidative stress response, which also contributes to cancer progression. Nrf2 activation reprograms the metabolic profile to promote the proliferation of cancer cells. A recent report suggested that K-ras- and Nrf2-active lung cancer cells are sensitive to glutamine depletion. This finding led to the recognition of glutaminase inhibitors as novel anticancer agents. In the current study, we used murine pancreatic cancer tissues driven by mutant K-ras and p53 to establish cell lines expressing constitutively activated Nrf2. Genetic or pharmacological Nrf2 activation in cells via Keap1 deletion or Nrf2 activation sensitized cells to glutaminase inhibition. This phenomenon was confirmed to be dependent on K-ras activation in human pancreatic cancer cell lines harboring mutant K-ras, i.e., Panc-1 and MiaPaCa-2 in response to DEM pretreatment. This phenomenon was not observed in BxPC3 cells harboring wildtype K-ras. These results indicate the possibility of employing Nrf2 activation and glutaminase inhibition as novel therapeutic interventions for K-ras mutant pancreatic cancers.

Keywords: BPTES; CB-839; Keap1; Nrf2; glutaminase.

Figure 1

Expression of Nrf2 and Keap1 in KPC, KPCN, K0N1, and K0N0 lines. Histone H3 and tubulin were used as loading controls for the proteins present in nuclear and cytosolic fractions, respectively.

Figure 2

Real-time RT-PCR for checking the expression of Gstm1 (A) and Cytokeratin 19 (B) in K0N1 and K0N0 cell lines (N = 4). ** indicates p < 0.01 by the Tukey–Kramer method. (C) BrdU assay in K0N1 and K0N0 cell lines following culture for 24 h in normal growth medium (N = 6). The error bars show standard deviations.

Figure 3

MTT viability assay for K0N1 and K0N0 cell lines after treatment with CB-839 (upper panel) and BPTES (lower panel) (N = 6, 5000 cells/well, 48 h). * and ** indicate p < 0.05 and p < 0.01 by the Tukey–Kramer method, respectively. The error bars show standard deviations.

Figure 4

Induction of Nrf2 and sensitization to CB-839 by DEM treatment in KPC lines. (A) Nuclear accumulation of Nrf2 and induction of Nqo1 expression in KPC cell lines after DEM treatment. Histone H3 and tubulin are displayed as loading controls for proteins present in the nuclear and cytosolic fractions, respectively. (B) MTT assay for analyzing the viability of KPC cell lines after CB-839 treatment (N = 6, 5000 cells/well, 48 h; with or without DEM pretreatment (100 μM, 24 h)). ** indicates p < 0.01 by the Tukey–Kramer method. The error bars show standard deviations.

Figure 5

Induction of Nrf2 and sensitization to CB-839 by DEM treatment in human pancreatic cancer cell lines. (A) Nuclear accumulation of Nrf2 in BxPC3, Panc-1, and MiaPaCa-2 cells after DEM treatment. Histone H3 is used as the loading control. (B) MTT assay for checking the viability of BxPC3, Panc-1 and MiaPaCa-2 after CB-839 treatment (N = 6, 5000 cells/well, 48 h; with or without DEM pretreatment (100 μM, 24 h)). ** indicates p < 0.01 by the Tukey–Kramer method. The error bars show standard deviations.