Cancer Cells Expressing Oncogenic Rat Sarcoma Show Drug-Addiction Toward Epidermal Growth Factor Receptor Antibodies Mediated by Sustained MAPK Signaling

Abstract

Epidermal growth factor receptor (EGFR) antibodies may have detrimental effects in patients with metastatic colorectal cancer expressing oncogenic Rat sarcoma (RAS). Since a significant number of patients acquire RAS-mediated resistance during EGFR-directed treatment, understanding the molecular mechanism underlying these antibody-mediated tumor-promoting effects is of relevance to design more resistance-preventive treatment approaches. To test this, we set up a Ba/F3 cellular model system transformed to EGFR/RAS dependency to be able to study proliferation, RAS activity as well as MAPK signaling upon inhibition of wild-type RAS isoforms by therapeutic EGFR antibodies. Here, we show that the EGFR antibodies cetuximab and panitumumab induce paradoxical stimulation and enhance proliferation in cells expressing oncogenic RAS (KRAS G12V). These experiments clearly showed that the stimulatory effect is a direct result of the antibody-EGFR interaction leading to prolonged mitogen-activated protein-Kinase (MAPK) signaling. The effect was also induced by antibody-chemotherapy combinations but always depended on simultaneous low-level ligand-dependent EGFR pathway activation. Moreover, we observed significant growth retardation of RAS mutant cells after antibody withdrawal compatible with a drug-addiction phenotype. Our data suggests that EGFR antibodies paradoxically sustain MAPK signaling downstream of oncogenic RAS thereby driving proliferation of RAS mutant tumors or tumor subclones. The observed drug-addiction encourages fixed-duration or liquid-biopsy-guided drug holiday concepts to preventively clear RAS mutant subclones selected under EGFR-directed therapeutic pressure.

Keywords: EGFR antibodies; MAPK signaling; acquired resistance; colorectal cancer; oncogenic RAS.

Figure 1

Set-up of hEGFR- and hKRAS-transducible hEGF-dependent Ba/F3 cellular model system. (A) Schematic overview of cellular Ba/F3 model system. Different Ba/F3 model cell lines were transduced with LeGO vectors to express hEGFR wild-type or G465R mutant genes (cDNAs) in conjunction with eGFP and/or the hKRAS wild-type or G12V mutant in combination with mCherry. (B) Growth properties of different Ba/F3 cell lines in absence and presence of hEGF. Proliferation of hEGFR wt, hEGFR wt / hKRAS wt (high), hEGFR wt / hKRAS G12V, hEGFR G465R, hKRAS G12V, and hEGFR G465R / hKRAS G12V transduced Ba/F3 cells was assessed in the absence and presence of hEGF. Cells were seeded in triplicate at equal densities and the average number of viable cells was measured after 72 h by trypan blue exclusion using Vi-CELL Cell Viability Analyzer. Experiments were performed three times with (n = 3). Results of one representative experiment are represented as mean ± SD. Statistical significance was calculated using 2-way ANOVA followed by a Sidak post-hoc test for multiple comparison (***p < 0.001).

Figure 2

Cetuximab and panitumumab drive proliferation of RAS mutant cells in the presence of EGF. (A) hEGFR wt / hKRAS G12V are paradoxically stimulated by EGFR-targeting antibody in the presence of hEGF. hEGFR wt, hEGFR wt / hKRAS G12V, or hEGFR G465R transduced Ba/F3 cells were seeded in triplicates at equal densities and treated with hEGF in combination with an EGFR-targeting antibody as indicated. Proliferation was assessed by counting the average number of viable cells every 24 h for 7 days using Vi-CELL Cell Viability Analyzer after trypan blue staining. For each treatment, data is expressed as the fraction of maximal cell count at 168 h normalized to the hEGF-stimulated control. Data is represented as mean ± SD with (n = 6). Statistical significance was calculated by t-test (***p < 0.001; ns, not significant). (B) Stimulatory antibody effect is present only upon engagement of the hEGFR signaling pathway by hEGF in hEGFR wt / hKRAS G12V Ba/F3 cells. Proliferation of hEGFR wt / hKRAS G12V transduced Ba/F3 cells was assessed in the absence or presence of hEGF plus cetuximab or panitumumab as indicated. Cells were seeded in triplicates at equal densities and the average number of viable cells was measured by trypan blue staining every 24 h for 7 days using Vi-CELL Cell Viability Analyzer. For each treatment, data is expressed as the fraction of maximal cell count at 168 h normalized to its respective control. Experiments were performed two times and results are represented as mean ± SD with (n = 6). Statistical significance was calculated using unpaired student's t-test (***p < 0.001; *p < 0.05; ns: not significant). (C) Stimulatory antibody effect persists in the presence of oxaliplatinum and irinotecan. hEGFR wt / hKRAS G12V transduced Ba/F3 cells were treated with hEGF, cetuximab and oxaliplatinum or irinotecan (IC50 dosing) as indicated. Cells were seeded in triplicates at equal densities and the average number of viable cells was measured by trypan blue staining every 24 h for 7 days using Vi-CELL Cell Viability Analyzer. For each treatment, data is expressed as viable cell count at 168 h. The experiment was performed in triplicates and results are represented as means ± SD with (n = 3). Statistical significance was calculated using 2-way ANOVA followed by a Tukey post-hoc test for multiple comparison (**p < 0.001).

Figure 3

EGFR antibodies lead to drug-addiction phenotype of RAS mutant cells. hEGFR wt / hKRAS G12V Ba/F3 cells show restricted growth after antibody withdrawal. hEGFR wt / hKRAS G12V and hEGFR G465R / hKRAS G12V transduced Ba/F3 cells were treated with hEGF and cetuximab as indicated. Cells were seeded in triplicates at equal densities and the average number of viable cells was measured by trypan blue staining every 24 h for 7 days using Vi-CELL Cell Viability Analyzer. After 7 days, the therapeutic antibody was withdrawn and cells were washed three times with phospho-buffered saline (PBS). Cells were set back to the initial cell count of one million cells and incubated in hEGF-containing media for another week yet without cetuximab. For each treatment, data is expressed as the fraction of maximal cell count at 168 h normalized the hEGF-stimulated control. Results are represented as mean ± SD with (n = 3). Statistical significance was calculated using 2-way ANOVA followed by a Sidak post-hoc test for multiple comparison (***p < 0.001; ns, not significant).

Figure 4

Paradoxical growth stimulation of RAS mutant cells by cetuximab requires EGFR binding. Proliferation of hEGFR wt, hEGFR wt / hKRAS G12V, hEGFR G465R, hKRAS G12V, and hEGFR G465R / hKRAS G12V transduced Ba/F3 cells was assessed in the absence of hEGF (control) or in the presence of hEGF, hEGF and cetuximab or cetuximab alone. Cells were seeded in triplicate at equal densities and cell viability was assessed by WST-8 assay. For each treatment, data are expressed as viable cell count at 168 h after initiation. Data is represented as mean ± SD with (n = 3). Statistical significance was calculated using 2-way ANOVA followed by a Tukey post-hoc test for multiple comparison (***p < 0.001).

Figure 5

Cetuximab treatment sustains MAPK signaling in RAS mutant cells. (A) Short-term effects of cetuximab treatment on RAS_GTP loading and ERK phosphorylation in RAS mutated and non-mutated cells. hEGFR wt or hEGFR wt / hKRAS G12V transduced Ba/F3 cells were cultured with or without hEGF (5 ng/ml) and/or cetuximab (5 μg/ml) for 2 h. Protein from whole cell lysates was either directly subjected to Western Blot for total RAS, ERK1/2, phospho-ERK1/2, and GAPDH as a loading control or RAS_GTP was precipitated by GTP pulldown followed by SDS-PAGE before Western Blot was performed. The Odyssey CLx Infrared Imaging System was used for signal detection and quantification. One representative experiment out of three is shown. (B) Long-term effects of EGF and cetuximab treatment on ERK phosphorylation in RAS mutated cells. Experiments were performed as in (A) described, except cells were treated for 2, 6, 24, or 96 h before protein was obtained. Further, only ERK1/2 phosphorylation (pERK1/2) of hEGFR wt / hKRAS G12V transduced Ba/F3 cells treated with hEGF (5 ng/ml) alone or together with cetuximab (5 μg/ml) is shown. Western Blot of one representative experiment is shown and pooled data from three to four experiments was quantified using Fiji version 2.0.0-rc-46/1.5 g (ImageJ, Maryland, USA) and shown in the graph. Results are represented as mean ± SEM with (n = 3–4).

Figure 6

Schematic overview of working hypothesis. Strong ligand-mediated pathway activation of RAS mutated cells attenuates cellular growth and proliferation via RAS-isoform mediated negative feedback inhibition (left graphics). Cetuximab is able to limit ligand-receptor interaction and thereby prevents feedback inhibition on oncogenic RAS resulting in enhanced cellular growth and proliferation (right graphics).