Additional file 1: of Pan-RAF and MEK vertical inhibition enhances therapeutic response in non-V600 BRAF mutant cells

Figure S1. Dose–response curves for sorafenib without or with the indicated selumetinib concentrations. Viability was measured by SRB assay after 10 days drug exposure and normalized to untreated controls (mean values +/− SEM). The respective combination indices (CI) were calculated by CompuSyn Software and are shown in Fig. 1c. (EPS 4764 kb

Additional file 2: of Pan-RAF and MEK vertical inhibition enhances therapeutic response in non-V600 BRAF mutant cells

Figure S2. Analysis of cell cycle after treatment with selumetinib or sorafenib and combination. Cells were treated with selumetinib (50 nM), sorafenib (1 μM), alone or in combination for 48 h. C – control, Se – selumetinib, So – sorafenib, Se + So – selumetinib + sorafenib. (EPS 8097 kb

Cell migration after zoledronic acid treatment in melanoma cells.

<p>(<b>A-C</b>) Migrated distance as a function of time and (<b>D</b>) average migrated distance after zoledronic acid (ZA) treatment in melanoma cells measured by videomicroscopy. A profound and significant increase in migrated distance was found in all of the BRAF mutant cells. A modest but significant increase in migration was found in VM-47 triple wild-type and VM-15 NRAS mutant cells. Colors blue, red and green indicate BRAF, NRAS mutation and wild-type for these genes, respectively. Data shown as average ± SEM are from at least three independent measurements. Asterisks indicate significant difference of p < 0.05 from the respective control with unpaired two-tailed T test.</p

<i>In vivo</i> effects of zoledronic acid treatment.

<p>Effect of zoledronic acid (ZA) treatment using <i>in vivo</i> subcutaneous xenograft model of human melanoma cells in SCID mice (<b>A, C, E</b>). ZA treatment failed to show effects in the subcutaneous growth of melanoma cells with either mutation. (<b>B, D, F</b>) Effect of ZA treatment using <i>in vivo</i> spleen liver colonization model of human melanoma cells in SCID mice. ZA did not inhibit the primary tumor or metastatic growth of melanoma cells. Data shown as average ± SEM.</p

Activation of downstream elements of the RAS/RAF pathway in melanoma cells after zoledronic acid treatment.

<p>(<b>A</b>) Representative blots of the effect of 48hs zoledronic acid (ZA) treatment on the activation of Erk1/2 and S6. (<b>B</b>) Quantification of the effect of ZA treatment on the activation of Erk1/2. Treatment with ZA resulted in robust increase in the phosphorylation of Erk1/2 in MEWO and M24met cells. (<b>C</b>) Quantification of the effect of ZA treatment on the activation of S6. After the treatment with ZA, decreased activation of S6 proteins was found only in NRAS mutant M24met and VM-15 cells. Colors blue, red and green indicate BRAF, NRAS mutation and wild-type for these genes, respectively. (C = control; ZA = zoledronic acid).</p

Cell viability, clonogenic growth and apoptosis in melanoma cells after zoledronic acid treatment.

<p>(<b>A</b>) Dose-response analysis of cell viability of human melanoma cell lines with different mutations after 72hs treatment with ZA measured by SRB assay; Most pronounced reduction in cell viability was observed in two NRAS mutant lines (<b>B</b>) Long-term effect of 10 days of 5 μM ZA treatment on clonogenic growth. Resistance was found in BRAF mutant and PTEN wild-type cells. Of note, NRAS and BRAF double mutant cells demonstrated intermediate sensitivity (<b>C</b>) Apoptosis induction after 25μM ZA treatment demonstrated by the proportion of TUNEL positive cells and (<b>D</b>) apoptosis induction of 72hs ZA treatment evaluated by the immunoblot of cleaved PARP. Limited apoptosis induction was found in BRAF mutant and PTEN wild-type cells and in the MDM2 over expressing WM239 cells. Colors green, red, blue, and dark-blue indicate triple wild-type, NRAS, BRAF, and BRAF mutant/PTEN-null mutational status of the cells, respectively. Data shown as average ± SEM are from at least 5 repeats. Asterisks indicate the lowest concentration of ZA treatment resulting in a significant difference with p < 0.05 from control by ANOVA and Dunnett’s post hoc test in the cell viability assay. Data shown as average ± SEM are from at least 3 measurements. Asterisks indicate significant difference of p < 0.05 between given mutational group and the BRAF mutant PTEN wild-type group by Kruskal-Wallis and Dunn’s post hoc test in the clonogenic assay, and p < 0.05 difference from the respective control by unpaired two tailed T test in the apoptosis assay. (C = control; Z = zoledronic acid).</p

EPOR is expressed in human lung ADC cell lines but exogenous rHuEPOα does not modify ADC cell proliferation in vitro.

<p>(A) Real-time qRT-PCR demonstrating the expression of EPOR mRNA in human lung ADC cell lines and K562 and HUVEC cells as control. The highest EPOR expression level was detected in the H1975 ADC cell line. H1975 (B), H1650 (C) and H358 (D) cells were treated with rHuEPOα at different concentrations (1, 3 IU/ml) with or without gemcitabine (1, 10 µg/ml). Cell numbers were estimated at 48 hours by sulforhodamine B colorimetric assay. Although gemcitabine significantly decreased the proliferation of ADC cells (<i>p</i> < 0.001), rHuEPO treatment (either alone or in combination with gemcitabine) did not modify ADC cell proliferation in vitro. </p

Low EPOR expression is associated with poor prognosis in advanced stage human pulmonary adenocarcinoma.

<p>(A) Kaplan-Meier curves for the overall survival of the entire patient population with stage III-IV ADC (n = 43), according to high and low EPOR expression as determined by the ratio of tumor and normal bronchoscopy brush EPOR mRNA expressions based on quantitative real-time PCR. </p

Effect of rHuEPOα and gemcitabine treatments on the proliferation of endothelial and tumor cells in H1975 xenograft tumors.

<p>Representative immunofluorescent images of tumors from control (A) and rHuEPOα-treated (B) mice. Tumor sections are stained for the endothelial marker, CD31 (green), the proliferation-associated marker, BrdU (red) and for TOTO-3 (blue) highlighting EC as well as tumor cell nuclei. Arrows in (B) point at proliferating endothelial cells. (C) Labeling index of tumor and endothelial cells in 33-day-old rHuEPOα-treated or control H1975 tumors. *<i>p</i> = 0.021, versus controls; **<i>p</i> < 0.001, versus controls. </p