Suppression of the HBP Function Increases Pancreatic Cancer Cell Sensitivity to a Pan-RAS Inhibitor

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death and the search for a resolutive therapy is still a challenge. Since KRAS is commonly mutated in PDAC and is one of the main drivers of PDAC progression, its inhibition should be a key strategy for treatment, especially considering the recent development of specific KRAS inhibitors. Nevertheless, the effects of KRAS inhibition can be increased through the co-inhibition of other nodes important for cancer development. One of them could be the hexosamine biosynthetic pathway (HBP), whose enhancement is considered fundamental for PDAC. Here, we demonstrate that PDAC cells expressing oncogenic KRAS, owing to an increase in the HBP flux, become strongly reliant on HBP for both proliferation and survival. In particular, upon treatment with two different compounds, 2-deoxyglucose and FR054, inhibiting both HBP and protein N-glycosylation, these cells undergo apoptosis significantly more than PDAC cells expressing wild-type KRAS. Importantly, we also show that the combined treatment between FR054 and the pan-RAS inhibitor BI-2852 has an additive negative effect on cell proliferation and survival by means of the suppression of both Akt activity and cyclin D1 expression. Thus, co-inhibition of HBP and oncogenic RAS may represent a novel therapy for PDAC patients.

Keywords: KRAS; KRAS inhibitors; PDAC; cancer treatment; glycosylations; hexosamine biosynthetic pathway.

Figure 1

KRASmut MIA PaCa-2 cells are more glycolytic than KRASwt BxPC-3. (A) The extracellular acidification rate (ECAR) of MIA PaCa-2 and BxPC-3 cells throughout a glycotest assay was measured using the Seahorse XF24 Analyzer. (B) The glucose consumption and lactate production were calculated measuring the amount of glucose and lactate in cell media of MIA PaCa-2 and BxPC-3. (C) MIA PaCa-2 and BxPC-3 cells were cultured in presence of the glycolysis inhibitor oxamate (OX) and counted at the indicated time points using the Trypan Blue stain. (D) The oxygen consumption rate (OCR) of MIA PaCa-2 and BxPC-3 cells throughout a mitostress test was measured using Seahorse XF24 Analyzer. (E) The production rate of the ATP due to the glycolysis (ATP Glyco) and the mitochondrial respiration (ATP Mito) was evaluated in MIA PaCa-2 and BxPC-3 cells by performing the specific assay with the Seahorse XFe96 Analyzer. (F) The XF Energy Map was determined using the Seahorse profiles. All data represent the mean ± SEM of at least three independent experiments. * p < 0.05, *** p < 0.001 (Student’s t-test), MIA PaCa-2 vs. BxPC-3.

Figure 2

KRASmut MIA PaCa-2 cells are more dependent on the hexosamine biosynthetic pathway (HBP) and glycosylation for their survival than KRASwt BxPC-3. (A) Protein O-GlcNAc and OGT expression were detected in total cell extracts from MIA PaCa-2 and BxPC-3 cells at 24, 48, and 72 h of culture. The expression of EIF2α was used for signal normalization. On the left, a representative image is shown, while the band intensity quantification of at least three independent blots is displayed in the histograms on the right. (B) MIA PaCa-2 and BxPC-3 cells were cultured in presence of different concentrations of 2-DG −/+ 1 mM mannose (MAN) and counted after 72 h using the Trypan Blue stain. (C) Representative microscopy images of MIA PaCa-2 and BxPC-3 cells upon 72 h-treatment with 2.5 mM 2-DG −/+ 1 mM MAN. All data represent the mean ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t-test), −MAN vs. +MAN in panel (B).

Figure 3

KRASmut MIA PaCa-2 cells are more sensitive than KRASwt BxPC-3 to the alteration of the N-glycosylations and the resulting endoplasmic reticulum (ER) stress. (A–D) Protein N-glycosylations were detected in viable MIA PaCa-2 (A,B) and BxPC-3 (C,D) cells after 72 h treatment with 2.5 mM 2-DG −/+ 1 mM mannose (MAN). In particular, tri-/tetra-branched N-glycans were recognized by FITC-conjugated PHA-L. The quantification of the mean fluorescence of independent experiments is displayed in the histograms (A,B), while representative flow cytometric profiles are shown in panels (C,D). (E,F) The expression of the UPR markers was analyzed through Western blot in MIA PaCa-2 (E) and BxPC-3 (F) cells treated with 2.5 mM 2-DG −/+ 1 mM MAN. Actin was used for signal normalization. The images are representative of at least three independent blots. The quantification of the EIF2α phosphorylation and Cleaved Caspase 3, considering all experiments performed, is displayed in the histograms on the bottom. All data represent the mean ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 (One-way ANOVA), untreated vs. treated where not specifically indicated.

Figure 4

KRASmut MIA PaCa-2 and PANC-1 cells are more sensitive to the HBP inhibition mediated by FR054 than KRASwt BxPC-3. (A) Cell viability of BxPC-3, MIA PaCa-2 and PANC-1 cells treated for 48 h with different concentrations of FR054 was detected by viable count using Trypan Blue stain. (B,C) Cell death of BxPC-3, MIA PaCa-2 and PANC-1 treated with 0.5 mM FR054 was evaluated by viable count (B) and by using flow cytometric analysis of Annexin V-FITC and propidium iodide staining (C). Representative profiles of the flow cytometric analysis are shown on the right of panel (C). All data represent the mean ± SEM of at least three independent experiments. * p < 0.05 and ** p < 0.01 (Student’s t-test in panel (A); one-way ANOVA in panels (B,C)), MIA PaCa-2 and Panc-1 vs. BxPC-3 (black asterisks) and untreated vs. treated (grey asterisks) where not specifically indicated.

Figure 5

In KRASmut MIA PaCa-2 and PANC-1 cells, FR054 and the pan-RAS inhibitor BI-2852 present additive negative effects on cell proliferation and survival. (A,B) Cell viability of MIA-PaCa-2 and PANC-1, treated for 72 h with FR054 (350 µM for MIA-PaCa-2, 500 µM for PANC-1) and different doses of RAS inhibitor BI-2852 (RASi), was detected through MTT test. (C–G) MIA PaCa-2 and PANC-1 were treated with FR054 and 50 µM BI-2852 for 72 h. (C–E) Cell death was evaluated by viable count using Trypan Blue stain (C,E) and by using flow cytometric analysis of annexin V-FITC and propidium iodide staining (D,F). Representative profiles of the flow cytometric analysis are shown on the right of the panels (D,F). (G) The protein expression was analyzed through Western blotting. Actin was used for signal normalization. All data represent the mean ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 (One-way ANOVA), untreated vs. treated where not specifically indicated.

Figure 6

Oncogenic KRAS expression in NIH3T3 cells induces sensitivity to HBP and RAS inhibition. (A) Protein O-GlcNAc levels as well as the expression of the HBP proteins were analyzed through Western blotting in normal (N), transformed (T) and reverted (R) NIH3T3 cells. As a loading control, Ponceau staining and actin expression were used, respectively. Representative images are shown, while the band intensity quantification of at least two independent blots is displayed in the histograms. (B–F) Cell number (C,E) and cell death (B,D,F) of the NIH3T3 cells were evaluated through Trypan Blue assay upon 72 h treatment with 2.5 mM 2-DG −/+ 1 mM mannose (B), with different concentrations of FR054 (C,D), and with 350 µM FR054 −/+ 50 µM BI-2852 (E,F). Data represent the mean ± SEM of at least two independent experiments. * p < 0.05, ** p < 0.01 (one-way ANOVA), untreated vs. treated and T vs. N or R (see colored asterisks), where not specifically indicated.