(µM) Erlotinib

In document Novel therapies in non-small cell lung cancer (Page 158-163)

MATERIALS & METHODS Drugs & chemicals

IC 50 (µM) Erlotinib

Sorafenib 2.880.35 1.100.22

6.361.00 0.820.13

10.170.79 0.940.27

0.500.17 0.880.22

9.011.80 0.960.17

20.122.21 2.070.64

0.100.02 1.120.23 Mutations

EGFR

k-Ras Wt

Mut(G12S) Wt

Mut(G12C) Wt

Mut(Q61H) Wt

Wt Wt

Wt Mut(T790M)

Wt Mut(L858R) Wt Polymorphisms

rs#1130233

rs#776746 GA

GG GG

GG GA

GG GA

GG GG

GG GG

GG GG

GG Expressiona

EGFR

RKIP 684.45

12.35 57.93

22.12 79.50

18.90 456.71

15.24 95.28

13.20 45.23

4.58 25.12 8.93

aMean mRNA expression values calculated in comparison with standard curves and with respect to the respective expression values of the housekeeping gene beta-actin, SE are less than 20%. Data were reported with 2 decimals, given the accuracy of the method used.

Pharmacological interaction

Since the CI method recommends a ratio of concentrations at which drugs are equipotent, combination studies were performed using fixed ratios with IC50 values for erlotinib and sorafenib in all the NSCLC cells.

Representative growth inhibition curves for A549 cells are shown in Fig.1A. Multiple drug-effect analysis revealed synergistic/additive effects at the more relevant FA values (≥50%). The average CI values for all the combinations in the seven NSCLC cell lines are summarized in Fig.1B. To explore the mechanisms underlying these synergistic drug interactions, we performed several biochemical analyses, as detailed below. Cell cycle and apoptosis studies were performed in 6 cell lines (A549, SW1573, H460, H292, H3255 and H1975), while PCR, ELISA, Western blot, LC-MS/MS, and peptide arrays were performed on A549 and H1975 cells.

Cell cycle perturbations and induction of apoptosis

Flow cytometric DNA analysis was performed to evaluate the effect of erlotinib, sorafenib and their combination on cell cycle distribution and to determine whether or not their cell cycle alterations might provide clues to optimise drug scheduling. Both targeted agents were able to affect the cell cycle parameters of the NSCLC cell lines (Table 2). In particular, both erlotinib and sorafenib caused a 1.2-1.4-fold increase in the population of cells in the G1-phase. The treatment with the erlotinib/sorafenib combination resulted in a

similar increase in the percentage of cells in the G1-phase. Conversely, after erlotinib, sorafenib and their combination, we observed slight variations of the S-phase and a reduction of the G2/M-phase cell population, which was most pronounced in H3255 cells (e.g., 3.3-fold).

Figure 1. Cytotoxicity and pharmacological interaction of erlotinib and sorafenib. (A) Representative curves of growth inhibitory effects of erlotinib, sorafenib, and their simultaneous 72-hour exposure. (B), Mean CI values of erlotinib-sorafenib combination in the panel of NSCLC cells. CI values at FA of 0.5, 0.75 and 0.9 were averaged for each experiment, and this value was used to calculate the mean between experiments, as described in the Materials and Methods section. Points and columns, mean values obtained from three independent experiments; bars, SE.

All treatments induced cell death, as shown by the presence of a cell population with sub-G1 DNA content in the FACS analysis. Similar results were observed by fluorescence microscopy analysis after bisbenzimide staining (Figure 2). Cells exposed to erlotinib, sorafenib and their combinations, at the IC50s, presented typical apoptotic morphology with cell shrinkage, nuclear condensation and fragmentation, and rupture into apoptotic bodies. Erlotinib slightly increased AI with respect to controls in A549, SW1573, H460 and H1975 cells, while higher AI values were observed in the more sensitive cell lines (6.5% and 14.2% in H292 and H3255 cells, respectively). Sorafenib induced more apoptosis than erlotinib, with AI values ranging from 6.5% to 15.6% in H1975 and H292 cells, respectively. The combinations of the two drugs additionally increased the AI, up to 28.0% in H3255 cells, with a significant induction of apoptosis when compared with both controls and erlotinib-treated cells in all cell lines.

Modulation of EGFR, ERK1/2 and Akt phosphorylation detected by ELISA

As expected, erlotinib induced a significant suppression of EGF-induced phosphorylation of EGFR at the tyrosine residue pY1173 in all the NSCLC cell lines, with percentages of reduction of EGFR phosphorylated protein ranging from -17.9 to -39.6% with respect to controls, in H1975 and A549 cells, respectively.

Conversely, sorafenib minimally affected pY1173-EGFR levels, varying from -5.6% to -6.2% in A549 and H1975 cells, respectively, while drug combinations reduced the phosphorylation status of EGFR, but to a lower extent than erlotinib alone.

EGFR signaling is transduced mainly through the Akt and ERK1/2 kinase pathways, and we investigated their phosphorylation status to determine their activity after drug treatment. Erlotinib and sorafenib resulted in an inhibition of pERK1/2 and pAkt in all the NSCLC cells. In particular, in both A549 and H1975 cells

pERK1/2 levels were potently (>50%) downregulated by sorafenib, while a lower degree of inhibition (about 25%) was detected after exposure to erlotinib. The drug combination cause a reduction of pERK1/2 more pronounced than the one observed with erlotinib or sorafenib alone in A549 cells (Fig. 3A), while drug combinations reduced the phosphorylation status of pERK1/2, to a similar extent than sorafenib alone in H1975 cells. Akt phosphorylation at the serine residue pS473 was significantly decreased (>50%) by erlotinib in A549 cells, whereas the inhibition was less efficient (about 30%) in H1975 cells. Akt phosphorylation was additionally reduced by the simultaneous combination of erlotinib and sorafenib, with a degree of inhibition up to -70.6% and -54.2 in A549 and H1975 cells, respectively.

Table 2. Cell cycle modulation and apoptotic index Cells Treatmenta G1

phase (%) S

phase (%) G2/M

phase (%) Sub-G1 phase (%)

A549

Control 59.92.4 21.11.4 18.91.4 1.20.2 Erlotinib 69.22.0 18.21.5 12.52.3 4.50.5* Sorafenib 67.94.3 20.13.2 12.01.5 11.11.5* Combination 67.55.0 21.33.6 11.21.6 18.62.2**

SW1573

Control 47.24.0 24.62.3 28.21.9 0.80.2 Erlotinib 55.06.6 23.19.0 21.92.2 4.81.0* Sorafenib 45.51.8 23.52.3 31.04.1 16.32.1* Combination 50.23.9 24.24.7 25.81.8 20.23.0**

H460

Control 41.83.0 25.02.0 33.21.3 1.30.3 Erlotinib 57.73.2 19.23.7 23.13.7 3.51.0* Sorafenib 51.73.7 25.01.9 23.32.8 12.21.8* Combination 61.03.9 20.12.4 18.91.8 21.93.1**

H292

Control 46.82.2 24.72.2 28.54.4 1.00.3 Erlotinib 62.76.1 19.71.9 17.65.0 6.51.0* Sorafenib 55.23.9 23.51.8 21.33.6 13.62.7* Combination 54.32.5 25.71.3 20.03.9 18.22.4**

H3255

Control 64.22.5 22.80.6 13.01.4 1.20.3 Erlotinib 75.96.4 18.71.2 5.41.3 14.22.7* Sorafenib 72.24.3 19.11.9 8.72.5 10.60.5* Combination 78.27.7 17.51.9 4.40.3 25.82.4**

H1975

Control 48.12.1 21.72.3 30.20.6 1.50.3 Erlotinib 56.01.7 20.22.2 23.82.5 4.50.6* Sorafenib 55.83.2 18.30.9 25.92.7 6.10.5* Combination 57.81.7 19.03.8 23.22.0 20.63.7**

*p<0.05 with respect to control cells, **p<0.05 with respect to pemetrexed-treated cells

Figure 2. Apoptotic index of erlotinib, sorafenib and their combination. The apoptotic index was calculated as the percentage of cells displaying apoptotic features compared to the number of counted cells. Points and columns, mean values obtained from three independent experiments; bars, SE. *Significantly different from controls (P<0.05);

**Significantly different from erlotinib (P<0.05)

Figure 3. Effects of erlotinib, sorafenib and their combination on phosphorylation of ERK1/2 and Akt, VEGF mRNA and protein, and RKIP mRNA expression. (A) Mean expression values of activated (ratio of phosphorylated compared to total) ERK and Akt, VEGF mRNA and protein, RKIP and E2F-1 mRNA. (B) Representative blot of at least two independent western blotting analyses of E2F-1 protein expression performed as described in the Material and Methods, in A549 (Bottom right panels) and H1975 cells (Bottom left panels). Points and columns, mean values obtained from three independent experiments; bars, SE. *Significantly different from controls (P<0.05);

**Significantly different from erlotinib (P<0.05)

Modulation of VEGF expression

Since erlotinib and sorafenib have been reported to have an impact on plasma VEGF levels, we evaluated the expression of VEGF levels in both cells and culture medium. However, sorafenib minimally affected VEGF mRNA levels and VEGF secretion into the medium in A549 and H1975 cells. Similarly, erlotinib and erlotinib-sorafenib combination hardly modulated VEGF in both A549 and H1975 cells (Fig.3A).

Modulation of RKIP and E2F-1 expression

To gain further insight into the molecular mechanisms underlying drug interaction, we examined alterations in the expression of RKIP, which is a key regulator of the RAS/RAF pathway, and E2F-1, which is an important nuclear target.

A549 and H1975 cells treated with erlotinib had a 1.5-fold and 2.4-fold increase in RKIP expression, respectively. In contrast, sorafenib slightly decreased RKIP levels in both cell lines. However, the erlotinib/sorafenib combination, not only prevented the sorafenib-induced decrease, but significantly increased RKIP mRNA expression, up to +67% and +40% in H1975 and A549 cells, respectively (Fig.3A).

Erlotinib, sorafenib and their combination significantly reduced E2F-1 mRNA in both A549 and H1975 cells, with expression levels of about 40-50% compared to untreated cells. Similarly, the erlotinib/sorafenib combination induced a significant decrease in E2F-1, with values ranging from -64 to -73% in H1975 and A549 cells, respectively (Fig.3A). E2F-1 expression was also studied at the protein level using Western blot analysis in both A549 and H1975 cells (Fig.3B). This analysis revealed a reduction in both erlotinib and erlotinib-sorafenib-treated cells, with barely detectable levels observed in protein extracts isolated from A549 cells exposed to erlotinib-sorafenib combination.

Peptide substrate arrays

Assuming that responses to tyrosine kinase inhibitors depend on specific receptor and protein signaling activities, kinase activity profiling is a promising approach to identify new potential markers and targets for

treatment. Therefore, we used high-throughput tyrosine kinase peptide substrate arrays for the identification of novel peptide substrate kinases or substrate kinases that were inhibited by erlotinib, sorafenib and their combination. In cell lysates, kinase activity profiling has been performed in absence and presence of 20 μM erlotinib, 25 μM sorafenib and their combination (20 μM erlotinib, 12.5 μM sorafenib). Data of Vini in treated cells were compared by t-test versus untreated cells. Using Bonferroni correction, we observed in the A549 extracts a total of 11 and 13 peptide substrates significantly inhibited by erlotinib and sorafenib, respectively, including 10 common targets. These peptides were also inhibited by the drug combination, which additionally inhibited other 14 peptides (Table 3). Similar results were observed in the H1975 cells (Table 3), and the Venn diagram (Fig. 4A) shows the common peptides which were inhibited in both cell lines.

With the aid of the KEGG and REACTOME databases, we were able to construct a provisional signal transduction scheme of some of the key kinases in NSCLC, including several kinases annotated as being able to phosphorylate the peptides emerging from the previous analyses (Figure 4B). This scheme showed kinase activity on peptides corresponding with phosphorylation site sequences from the Human Epidermal Growth Factor Receptor 2 (ERBB2), platelet-derived growth factor receptor beta (PDGFRB), vascular endothelial growth factor (VEGFR1), Fibroblast growth factor receptor 2 and 3 (FGFR2, FGFR3), and hepatocyte growth factor receptor (MET). In addition, we found associated downstream inhibition of Ras/MAPK pathways, focal adhesion kinase (FAK), and SRC kinase (correlated with LCK, and ZAP70) signaling (Fig.4B). Further studies are planned to validate these kinases also as potential druggable targets in erlotinib/sorafenib resistant cells. However, to evaluate some of our most interesting results we performed western blot analyses of CDK2 and SRC, showing a significant reduction of phosphorylation for both these kinases after exposure to erlotinib, sorafenib and their combination (Fig.4C). Finally, peptide substrate array-based kinase activity profiling was also performed using tumor tissue lysates of patients with NSCLC. These tumor tissues were already characterized within genetic analyses, and one tumor harboured the K-Ras G12D mutation, while the other carried EGFR exon 20 mutations.

The studies with the peptide substrate array demonstrated the feasibility of kinase profiling in patients’

tissues even when obtained by needle biopsy. However, the number of kinases significantly inhibited by erlotinib and sorafenib (i.e., 0 in patient#1, and 17 in patient#2) was lower than in the cells, and only 3 of these inhibited kinases (EFS, RASA1 and SRC8) were also significantly inhibited by erlotinib and sorafenib in the cells. These results suggest that these peptide array studies did not reveal response predictive profiles of kinase inhibition for the tested compounds in the two tumor samples from patients. However, further retrospective and prospective clinical studies are needed to demonstrate the capacity of this technique for the prediction of response to tyrosine kinase inhibitors.

LC-MS/MS measurement of erlotinib and sorafenib

Previous clinical trials reported significantly decreased steady-state plasma levels of the EGFR inhibitors erlotinib and gefinitib by the coadministration of sorafenib. Since the mechanism of this possible drug interaction is unclear, we tested the concentration of these drugs in cell pellets and medium and we evaluated whether sorafenib or erlotinib affected each other intracellular drug accumulation. However, no direct drug interaction was detected by LC-MS/MS measurement, and drug concentrations after exposure to

erlotinib or sorafenib alone were identical to concentrations detected after drug combination (data not shown).

In document Novel therapies in non-small cell lung cancer (Page 158-163)