Synthetic lethality screen identifies RPS6KA2 as modifier of epidermal growth factor receptor activity in pancreatic cancer

Abstract

Pancreatic cancer is characterized by a high degree of resistance to chemotherapy. Epidermal growth factor receptor (EGFR) inhibition using the small-molecule inhibitor erlotinib was shown to provide a small survival benefit in a subgroup of patients. To identify kinases whose inhibition acts synergistically with erlotinib, we employed a kinome-wide small-interfering RNA (siRNA)-based loss-of-function screen in the presence of erlotinib. Of 779 tested kinases, we identified several targets whose inhibition acted synergistically lethal with EGFR inhibition by erlotinib, among them the S6 kinase ribosomal protein S6 kinase 2 (RPS6KA2)/ribosomal S6 kinase 3. Activated RPS6KA2 was expressed in approximately 40% of 123 human pancreatic cancer tissues. RPS6KA2 was shown to act downstream of EGFR/RAS/mitogen-activated protein kinase kinase (MEK)/extracellular-signal regulated kinase (ERK) signaling and was activated by EGF independently of the presence of KRAS mutations. Knockdown of RPS6KA2 by siRNA led to increased apoptosis only in the presence of erlotinib, whereas RPS6KA2 activation or overexpression rescued from erlotinib- and gemcitabine-induced apoptosis. This effect was at least in part mediated by downstream activation of ribosomal protein S6. Genetic as well as pharmacological inhibition of RPS6KA2 by the inhibitor BI-D1870 acted synergistically with erlotinib. By applying this synergistic lethality screen using a kinome-wide RNA interference-library approach, we identified RPS6KA2 as potential drug target whose inhibition synergistically enhanced the effect of erlotinib on tumor cell survival. This kinase therefore represents a promising drug candidate suitable for the development of novel inhibitors for pancreatic cancer therapy.

Figure 1

Target validation of the RNAi screen results. Cell viability in BxPC-3 cells was determined in the presence of erlotinib after knockdown of the individual target genes using four independent silencing sequences or scrambled nonsilencing control siRNAs (siC).

Figure 2

RSK3 protein expression in pancreatic cancer. (A) RSK3 protein expression in various pancreatic cancer cell lines and the pancreatic ductal epithelial cell line HPDE, as assessed by Western blot analysis. (B) Representative immunohistochemical analysis of phospho-RSK3 (Thr³⁵⁶/Ser³⁶⁰) expression indicative of active RSK3 in human normal pancreatic tissue and pancreatic cancers. (C) RSK3 expression in murine pancreatic cancer tissue derived from the KPC mouse model, as assessed by immunohistochemistry using the phospho-RSK3 (Ser²²¹⁸) antibody.

Figure 3

RSK3 inhibition induces apoptosis synergistically with erlotinib, and RSK3 mediates resistance to apoptosis. (A) PaTu-8988t cells were transiently transfected with siRNA against RSK3 (siRSK3) or siC and treated with erlotinib or solvent DMSO. siRSK3 sensitizes PaTu-8988t cells to erlotinib treatment but is ineffective in the absence of erlotinib. (B) Clones stably overexpressing RSK3 or empty vector were incubated with gemcitabine and/or erlotinib. Apoptosis was determined by a DNA fragmentation assay. *P < .05 compared to siC (A) or Mock-transfected clones (B). Results are representative for three independent experiments.

Figure 4

RSK3 is activated by EGF/EGFR through the MEK/ERK signaling cascade and modulates rpS6 phosphorylation. Serum-starved cells were incubated with the EGFR inhibitor erlotinib, the MEK inhibitor UO126, or the PI3K inhibitor LY294002 for 2 hours before stimulation with EGF for 15 minutes. Phosphorylation status of ERK and RSK3 (A) as well as rpS6 (B) was determined using specific antibodies by Western blot analysis in PaTu-8988t cells harboring mutant KRAS. (C and D) BxPC-3 cells with wild-type KRAS (Mock) and BxPC-3 cells stably transfected with constitutively active KRAS (KRAS) were incubated with the indicated compounds as above. Phosphorylation status of ERK and RSK3 (C) as well as rpS6 (D) was determined using specific antibodies by Western blot analysis in BxPC-3 cells stably expressing wild-type (Mock) or mutant KRAS (KRAS). Note that the PI3K inhibitor LY294002 has a profound impact on rpS6 phosphorylation but not on RSK3 phosphorylation, suggesting that PI3K/mTOR signaling affects rpS6 phosphorylation independently of RSK3.

Figure 5

RSK3 phosphorylates rpS6. (A) PaTu-8988t cells were transiently transfected with siRSK3 or siC at two different siRNA concentrations (25 and 50 nM). Phosphorylated rpS6 and total rpS6 were detected with specific antibodies. (B) PaTu-8988t cells were treated with the RSK inhibitor BI-D1870 (5 µM) and/or erlotinib (10 µM) for the indicated time points. Phospho-rpS6 and total rpS6 were detected by specific antibodies.

Figure 6

Pharmacological inhibition of RSK3 acts synergistically with erlotinib. BxPC-3 (A) and PaTu-8988t (B) cells were treated with the RSK inhibitor BI-D1870 in the presence or absence of erlotinib for 48 hours. Cell viability of cells was assessed using MTT assays. Results are representative for three independent experiments. *P < .05 compared to erlotinib only and to erlotinib + BI-D (1 µM, A or 5 µM, B).

Figure 7

Synergistic effect of RSK inhibition by BI-D1870 and EGFR inhibition by erlotinib. (A) PaTu-8988t cells were treated with BI-D1870 (5 µM) and/or erlotinib (10 µM), and PARP cleavage was determined by Western blot analysis after 24 hours. (B) PaTu-8988t cells stably overexpressing RSK3 or empty vector (Mock) were treated with BI-D1870 and/or erlotinib, and apoptosis was determined by DNA fragmentation assays. *P < .05 compared to either Mock clone. Data are representative for three independent experiments.