KRAS(G12D)- and BRAF(V600E)-induced transformation of murine pancreatic epithelial cells requires MEK/ERK-stimulated IGF1R signaling

Abstract

Mutation of KRAS is a common initiating event in pancreatic ductal adenocarcinoma (PDAC). Yet, the specific roles of KRAS-stimulated signaling pathways in the transformation of pancreatic ductal epithelial cells (PDEC), putative cells of origin for PDAC, remain unclear. Here, we show that KRAS(G12D) and BRAF(V600E) enhance PDEC proliferation and increase survival after exposure to apoptotic stimuli in a manner dependent on MEK/ERK and PI3K/AKT signaling. Interestingly, we find that activation of PI3K/AKT signaling occurs downstream of MAP-ERK kinase (MEK), and is dependent on the autocrine activation of the insulin-like growth factor (IGF) receptor (IGF1R) by IGF2. Importantly, IGF1R inhibition impairs KRAS(G12D)- and BRAF(V600E)-induced survival, whereas ectopic IGF2 expression rescues KRAS(G12D)- and BRAF(V600E)-mediated survival downstream of MEK inhibition. Moreover, we show that KRAS(G12D)- and BRAF(V600E)-induced tumor formation in an orthotopic model requires IGF1R. Interestingly, we show that while individual inhibition of MEK or IGF1R does not sensitize PDAC cells to apoptosis, their concomitant inhibition reduces survival. Our findings identify a novel mechanism of PI3K/AKT activation downstream of activated KRAS, illustrate the importance of MEK/ERK, PI3K/AKT, and IGF1R signaling in pancreatic tumor initiation, and suggest potential therapeutic strategies for this malignancy.

Figure 1. KRAS^G12D and BRAF^V600E enhance proliferation and survival in pancreatic ductal epithelial cells (PDECs)

(A) Immunoblot confirming expression of ectopic FLAG epitope-tagged KRAS^G12D in Ink4a/Arf, Trp53 null PDECs infected with RCAS-KRAS^G12D. β-actin is used as a loading control. (B) Immunoblot confirming elevated expression of BRAF in Ink4a/Arf, Trp53 null PDECs infected with RCAS-BRAF^V600E. β-actin is used as a loading control. Numbers represent relative BRAF/β-actin levels in RCAS-BRAF^V600E-infected cells relative to RCAS-GFP infected controls. (C) Cell numbers of tumor suppressor wild type PDECs expressing KRAS^G12D (black bars), BRAF^V600E (grey bars), or GFP (white bars) at indicated time points after plating. Results are representative of at least two experiments. (D, E) Viability of tumor suppressor wild type PDECs expressing KRAS^G12D, BRAF^V600E, or GFP, treated with 100μM cycloheximide (D) or ultraviolet (UV) irradiation (E). White bars vehicle treated (or untreated) cells; black bars cycloheximide- or UV-treated cells. Values are normalized such that that viability of untreated cells is 1. Results are representative of at least two experiments. * p<0.01 compared to GFP expressing controls. (F) Cell numbers of Ink4a/Arf, Trp53 null PDECs expressing KRAS^G12D (black bars), BRAF^V600E (gray bars), or GFP (white bars) at indicated time points after plating. Results are representative of at least two experiments. (G) Viability of Ink4a/Arf, Trp53 double null PDECs expressing KRAS^G12D, BRAF^V600E, or GFP, treated with 100μM cycloheximide, as measured by trypan blue exclusion. * p<0.01 compared to GFP expressing controls. (H) Viability of Ink4a/Arf null PDECs expressing KRAS^G12D, BRAF^V600E, or GFP, following UV irradiation. White bars vehicle treated (or untreated) cells; black bars cycloheximide- or UV-treated cells. Values are normalized such that that viability of untreated cells is 1. Results are representative of at least two experiments. * p<0.01 compared to GFP expressing controls. All error bars, SD.

Figure 2. KRAS^G12D- and BRAF^V600E-induced survival in PDECs requires MEK/ERK and PI3K/AKT signaling

(A) Viability Ink4a/Arf, Trp53 double null of PDECs expressing KRAS^G12D, BRAF^V600E, SHH, or GFP, treated with DMSO, PD98059, or LY2900042 plus 100μM cycloheximide. * p<0.05 compared to DMSO treated cells of identical genotype. (B) Viability of Ink4a/Arf null PDECs expressing KRAS^G12D, BRAF^V600E, SHH, or GFP, treated with DMSO, PD98059, or LY2900042 plus UV irradiation. White bars vehicle treated (or untreated) cells; black bars cycloheximide or UV treated cells. Values are normalized such that viability of untreated cells is 1. Results are representative of at least two experiments. * p<0.05 compared to DMSO treated cells of identical genotype. (C) Immunoblot analysis of ERK (Thr202/Tyr204) and AKT (ser473) phosphorylation in serum starved KRAS^G12D, BRAF^V600E, and GFP expressing Ink4a/Arf, Trp53 null PDECs. Values indicate the ratio of phosphorylated AKT relative to total AKT as measured by densitometry and normalized such that GFP expressing PDECs have a ratio of 1. All error bars, SD.

Figure 3. AKT phosphorylation in KRAS^G12D- and BRAF^V600E-expressing PDECs depends on signaling through IGF1R

(A) Quantitative RT-PCR measurement of Igf2 mRNA in KRAS^G12D-, BRAF^V600E-, and GFP-expressing Ink4a/Arf, Trp53 null PDECs (black, gray, and white bars respectively). β-actin is used as an endogenous control. Ligand expression in GFP-expressing cells is normalized to 1. (B) Immunoblot analysis of IGF2 levels in serum starved KRAS^G12D-, BRAF^V600E-, and GFP-expressing Ink4a/Arf, Trp53 null PDECs. β-actin is used as a loading control. (C) Immunoblot analysis of IGF1R phosphorylation in serum starved KRAS^G12D-, BRAF^V600E-, and GFP-expressing Ink4a/Arf, Trp53 null PDECs. Total IGF1R levels are also shown. β-actin is used as a loading control. (D) Quantitative RT-PCR measurement of Igf2 mRNA in serum starved in KRAS^G12D-expressing Ink4a/Arf, Trp53 null PDECs treated with PD98059, LY294002, and AG1024. β-actin is used as an endogenous control. (E) Immunoblot analysis of ERK (Thr202/Tyr204) and AKT (ser473) phosphorylation in serum starved KRAS^G12D-, BRAF^V600E-, and GFP-expressing Ink4a/Arf, Trp53 null PDECs treated with PD98059, LY294002, and AG1024. (F) Immunoblot analysis of ERK (Thr202/Tyr204) and AKT (ser473) phosphorylation, as well as IGF1R expression in serum starved KRAS^G12D-, BRAF^V600E-, and GFP-expressing Ink4a/Arf, Trp53 null PDECs expressing an IGF1R-targeting shRNA. All error bars, SD.

Figure 4. KRAS^G12D- and BRAF^V600E-induced survival in PDECs requires IGF1R signaling

(A) Viability of Ink4a/Arf, Trp53 double null PDECs expressing KRAS^G12D, BRAF^V600E, SHH, or GFP treated with DMSO or AG1024, plus 100μM cycloheximide. * p<0.01 compared to DMSO treated cells of identical genotype. (B) Viability of Ink4a/Arf null PDECs expressing KRAS^G12D, BRAF^V600E, SHH, or GFP treated with DMSO or AG1024 plus UV irradiation as measured by trypan blue exclusion. White bars untreated cells, black bars UV treated cells. Values are normalized such that viability of untreated cells is 1. Results are representative of at least two experiments. * p<0.01 compared to DMSO treated cells of identical genotype. (C) Impact of IGF1R- and IR-targeting shRNAs on the viability of KRAS^G12D- and GFP-expressing Ink4a/Arf, Trp53 double null PDECs treated with 100μM cycloheximide. * p<0.05 compared to cells treated with control shRNA. (D) Viability of Ink4a/Arf, Trp53 null PDECs expressing KRAS^G12D, BRAF^V600E, or GFP, as well as ectopic IGF2 (or GFP as a control), following treatment with 100μM cycloheximide and the indicated inhibitors. White bars vehicle treated cells, black bars cycloheximide treated cells. Values are normalized such that viability of untreated cells is 1. Results are representative of at least two experiments. * p<0.05 compared to GFP expressing cells treated with PD98059. All error bars, SD.

Figure 5. Combined inhibition of MEK and IGF1R impairs pancreatic cancer cell survival

(A) Viability of the KRAS^G12D-expressing, Ink4a/Arf, Trp53 null murine pancreatic cancer cell line 170#3 following treatment with 100μM cycloheximide and the indicated inhibitors. White bars vehicle treated cells; black bars cycloheximide treated cells. Values are normalized such that that viability of untreated cells is 1. Results are representative of at least two experiments. * p<0.05 compared to DMSO treated cells. (B) Viability of the human Panc1 pancreatic cancer cell line following treatment with 100μM cycloheximide and the indicated inhibitors. White bars vehicle treated cells; black bars cycloheximide treated cells. Values are normalized such that that viability of untreated cells is 1. Results are representative of at least two experiments. * p<0.05 compared to DMSO treated cells. (C) Viability of the 170#3 cell line following treatment with 50nM gemcitabine and the indicated inhibitors. White bars vehicle treated cells; black bars gemcitabine treated cells. Values are normalized such that that viability of untreated cells is 1. Results are representative of at least two experiments. * p<0.05 compared to DMSO treated cells. All error bars, SD.