Analysis of EGFR downstream pathways in parental CALU-3 and HCT116 cells (WT) and in their TKI-resistant derivatives 8ERL-R, GEF-R, VAN-R).

<p>Western blotting analysis of EGFR and of down-stream signalling pathways in parental human CALU-3 and HCT116 cells (WT) and in their TKI-resistant derivatives (ERL-R, GEF-R, VAN-R). β-actin was included as a loading control.</p

Effects of treatment with sorafenib on the invasive, migratory and anchorage-independent colony forming capabilities of TKI-resistant CALU-3 and HCT116 cancer cells.

<p>Anchorage-independent growth (A), migration (B) and invasion (C), were evaluated in TKI-resistant CALU-3 and HCT116 derivatives (ERL-R, GEF-R, VAN-R) after treatment with the indicated concentrations of sorafenib. The results are the average ± SD of three independent experiments, each done in triplicate. Representative pictures are shown for the migration and invasion abilities of CALU-3 WT and Resistant cell lines.</p

IC50 for treatment with erlotinib, gefitinib or vandetanib in parental CALU-3 and HCT116 cell lines (WT) and their TKI-resistant derivatives (ERL-R, GEF-R, VAN-R).

<p>IC50 for treatment with erlotinib, gefitinib or vandetanib in parental CALU-3 and HCT116 cell lines (WT) and their TKI-resistant derivatives (ERL-R, GEF-R, VAN-R).</p

Antitumor activity of the sorafenib in parental and TKI-resistant CALU-3 xenografts.

<p>A, Parental (WT) CALU-3 cancer cells; B, GEF-R CALU-3 cancer cells; C, VAN-R CALU-3cancer cells; D, ERL-R CALU-3 cancer cells. Athymic nude mice were injected subcutaneously into the dorsal flank with 10⁷ cancer cells. After 7 to 10 days (average tumor size, 75 mm³), mice were treated as indicated in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028841#s4" target="_blank">Methods</a> for 5 weeks. Each treatment group consisted of 8 mice. Data represent the average (±SD). Student's <i>t</i> test was used to compare tumor sizes among different treatment groups at day 35 following the start of treatment. A, CALU-3 WT: sorafenib versus control (two-sided p<0.001); B, CALU-3 GEF-R: sorafenib versus control (two-sided p<0.001). C, CALU-3 VAN-R: sorafenib versus control (two-sided p<0.001); D, CALU-3 ERL-R: sorafenib versus control (two-sided p<0.001).</p

Growth inhibitory effects of treatment with sorafenib in parental and TKI-resistant CALU-3 and HCT116 cancer cells.

<p>MTT cell proliferation assays were performed in parental lung adenocarcinoma CALU-3 and colorectal cancer HCT116 cells. (WT) and in their TKI-resistant derivatives (ERL-R, GEF-R, VAN-R ), treated for three days with the indicated concentrations of sorafenib. Results represent the average (±SD) of three separate experiments, each performed in quadruplicate.</p

Western blotting analysis of CALU-3 and HCT116 cells TKI-resistant derivatives (ERL-R, GEF-R, VAN-R) following treatment with sorafenib.

<p>Western blotting analysis of C-RAF, B-RAF, MEK and MAPK activation following treatment with the indicated concentration of sorafenib of lung adenocarcinoma CALU-3 and colorectal cancer HCT116 cells TKI-resistant derivatives (ERL-R, GEF-R, VAN-R ). β-actin was included as a loading control.</p

Antitumor activity of the sorafenib in parental and TKI-resistant HCT116 xenografts.

<p>A, Parental (WT) HCT116 cancer cells; B, GEF-R HCT116 cancer cells; C, VAN-R HCT116 cancer cells; D, HCT116 cancer cells. Athymic nude mice were injected subcutaneously into the dorsal flank with 10⁷ cancer cells. After 7 to 10 days (average tumor size, 75 mm³), mice were treated as indicated in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028841#s4" target="_blank">Methods</a> for 5 weeks. Each treatment group consisted of 8 mice. Data represent the average (±SD). Student's <i>t</i> test was used to compare tumor sizes among different treatment groups at day 35 following the start of treatment. A, HCT116 WT: sorafenib versus control (two-sided p<0.001); B, HCT116: sorafenib versus control (two-sided p<0.001). C, HCT116 VAN-R: sorafenib versus control (two-sided p<0.001); D, HCT116 ERL-R: sorafenib versus control (two-sided p<0.001).</p

Supplementary Figure Legends from Primary and Acquired Resistance of Colorectal Cancer Cells to Anti-EGFR Antibodies Converge on MEK/ERK Pathway Activation and Can Be Overcome by Combined MEK/EGFR Inhibition

PDF file - 116KB</p

Supplementary Figures 1 - 3, Tables 1 - 3 from Primary and Acquired Resistance of Colorectal Cancer Cells to Anti-EGFR Antibodies Converge on MEK/ERK Pathway Activation and Can Be Overcome by Combined MEK/EGFR Inhibition

PDF file - 1325KB, Caption Supplementary Figure 1 A and 1B. The combined treatment of cetuximab and the selective MEK1/2 inhibitor BAY 86-9766 induce a synergistic growth inhibitory effects in CRC cell lines with primary and acquired resistance to cetuximab and an antagonistic effects in cetuximab-sensitive CRC cell lines. Caption Supplementary Figure 2. The BAY 86-9766 treatment, in CRC cancer cell lines with primary and acquired resistance to cetuximab, induce a dose dependent reduction of MAPK and MEK phosphorylation. Caption Supplementary Figure 3. The combined treatment of cetuximab and the selective MEK1/2 inhibitor BAY 86-9766 was well tolerated by mice, with no weight loss or other signs of acute or delayed toxicity. Caption Supplementary Table 1A and 1B. The panel of CRC cell lines have been treated with several concentrations of cetuximab and selective MEK1/2 inhibitors (BAY 86-9766, Selumetinib and Pimasertib), showing differential sensitivity to the drugs. Caption Supplementary Table 2. We have performed the experiments on a panel of eight human CRC cell lines having different mutation profiles in KRAS, NRAS, BRAF, PIK3CA and EGFR genes. Caption Supplementary Table 3. In the CRC cell lines with acquired and primary resistance to cetuximab, the treatment with the selective MEK1/2 inhibitor BAY 86-9766 determined synergistic growth inhibitory effects in combination with cetuximab.</p

Mutational status of the samples used for the quality assessment.

<p>* = samples chosen for the external quality assessment program: A1–A5: samples for the first round; B1–B5: samples for the second round.</p