Fig 1: Absence of ARAF kinase activity enables formation of metastasis in the lung and lymph nodes of nude mice.(A) Western blotting of A549 cells that were depleted for ARAF and reconstituted with either the empty vector (EV), WT ARAF (+ARAF), kinase-active (+DD), and kinase-deficient (R362H), respectively. Cells (30,000) were taken for Western blotting to evaluate ARAF (V5) overexpression and its effect on MAPK signaling or (B) mixed with 0.9% agarose and poured onto plates containing 1.5% agarose base for growing. Colonies were stained with crystal violet and photographed 14 days later. Quantification of colonies as fold change. Data are means ± SEM from three independent experiments. ***P < 0. 001, one-way ANOVA with post hoc t tests using a correction of alpha according to Bonferroni. (C) Mice were injected with 1 × 106 stably expressing luciferase control and ARAF-depleted A549 cells reconstituted with WT ARAF (control), EV, and kinase-deficient mutant (R362H), respectively (from left to right). Representative images are shown. In vivo bioluminescence imaging was done 14, 17, and 21 days after injection of the tumor cells via the tail vein. Images were captured 10 min after injection of 100 µl of d-Luciferin (40 mg/ml) (n = 6). (D) Scatterplots show the total counts of emitted photons in ROIs at 17 days. ROIs were assigned to lung, lymph nodes, abdomen, and bones according to the anatomy and overlay with a standard mouse (implemented in LivingImage). Data are means ± SEM from six mice. *P < 0. 05, one-way ANOVA with post hoc t tests using a correction of alpha according to Bonferroni. (E) Bar diagram shows the frequency of mice carrying metastases at the indicated sites 17 days after tumor cell injection. Chi-square P < 0.0001. ns, not significant.
Fig 2: ARAF regulates ligand-induced phosphorylation of AKT in an ERBB3-dependent manner.(A) A549 cells that were depleted for ARAF (+EV) and reconstituted with WT (+ARAF) and kinase-active (+ARAF-DD), respectively, were seeded in a 12-well dish at a density of 1 × 105 and stimulated 24 hours later with indicated concentrations of hNRG1 for 20 min. Phosphorylation levels of ERBB3 (Tyr1289), AKT (S473), and T308 were monitored with vinculin serving as a loading control. The phospho blots of ERBB3 were stripped and probed for checking total ERBB3 levels. (B) A549 cells that were depleted for ARAF(+EV) or reconstituted with WT ARAF were transiently transfected with siRNA targeting ERBB3 (siERBB3) or control siRNA (siCo) and stimulated with hNRG1 (100 ng/ml) for 20 min before lysates were subjected to Western blotting and probed for indicated proteins with vinculin serving as a loading control. (C) Quantification of pAKT levels (S473) presented in (B) from ARAF-depleted cells silenced for ERBB3 compared to siControl treated with hNRG1 (100 ng/ml), respectively. Fold change values were calculated after normalization with internal loading control (vinculin), and four independent experiments (n = 4) are represented as means ± SEM. *P < 0.05, t test. (D) Illustration of the dual role of ARAF in the regulation of the ERBB3-AKT signaling axis and metastasis. ARAF kinase controls the promoter activity of ERBB3 and thus its expression in a kinase-independent manner. ARAF partially suppresses NRG1-ERBB3-AKT activation in a kinase-dependent manner. Loss of ARAF promotes ERBB3-AKT signaling and metastasis in a cell type–dependent manner.
Fig 3: ARAF down-regulation results in anchorage-independent growth and lung colonization in mice.(A) Western blotting of A549 control and ARAF-depleted cells. Cells (50,000) were plated into 12-well dishes and lysed 24 hours thereafter to verify the knockdown of ARAF and concomitant decrease in ERK 1/2 phosphorylation. (B) Loss of ARAF promotes lung metastasis in nude mice after tail vein injection of A549 cells, carrying CMV promoter–driven luciferase gene expression. Representative bioluminescence images show luciferase activity in tumor-bearing mice. Mice were injected with 1 × 106 control and ARAF-depleted (shARAF) A549 cells that were stably transfected with the firefly luciferase gene. Bioluminescence images were captured 10 min after injection of d-Luciferin intraperitoneally, 2 weeks after injection of tumor cells via the tail vein. The control group comprised 13 mice, while the shARAF group comprised 11 mice. (C) Scatterplots show the total counts of emitted photons in regions of interest (ROIs), which were automatically identified, software-aided. Each scatter shows one ROI. (D) To assess group differences, total counts were compared with Mann-Whitney U tests. In addition, the numbers of mice bearing lung metastases were compared with chi-squared statistics (ncontrol = 13; nshARAF = 11; P = 0.039).
Fig 4: p-AKT and ERBB3 expression in lung NSCLCs.(A) IHC staining of p-AKT and ERBB3 in a human LUAD tumor sample that showed a negative staining for ARAF (H-score < 50). Staining was performed using TMAs as described in Materials and Methods. Arrowheads show weak cytoplasmic stainings, and the open arrowhead highlights weak membranous staining. Shown TMA is representative for patient samples that showed a negative staining of ARAF (H-score = 40), ERBB3 (H-score = 10), and p-AKT (H-score = 0). (B) Same as in (A), but with a human LUSC TMA, which is representative for patients with negative ARAF staining (H-score = 5), but positive ERBB3 (H-score = 74) and p-AKT (H-score = 60) staining. (C) The pAKT H-score was calculated as described in Materials and Methods based on the percentages of weakly, moderately, and strongly stained tumor cells in primary tumor samples (n = 45) and normal adjacent epithelial cells, pneumocytes (n = 10). Results are presented as means ± SD. An unequal variance Welch t test was performed (P = 0.7355). (D) The pAKT H-scores are presented in correlation to the ARAF H-scores that were below 50, thus representing tumor samples with an ARAF-negative staining. (E and F) Same as in (C) and (D), but H-scores of ERBB3 were analyzed. An unequal variance Welch t test was performed, indicating a significant difference in the ERBB3 H-scores between normal and tumor samples (***P < 0.0001).
Fig 5: ARAF expression in lung NSCLCs.(A) IHC staining of ARAF and analyses of NSCLC tumor and adjacent normal tissue were performed using tissue microarrays (TMAs) (as described in Materials and Methods). Shown are representative images of no anti-ARAF staining of tumor cells (a, H-score = 0), of an ARAF staining of a LUSC tissue sample (b, H-score = 20), and of a LUAD tissue sample (c, H-score = 80). The arrow highlights anthracosis, and the arrowheads show weak cytoplasmic anti-ARAF staining of tumor cells. (B) The ARAF H-score was calculated on the basis of the percentages of weakly, moderately, and strongly stained tumor cells in primary tumor samples (n = 84) and normal adjacent epithelial cells (n = 13) as described in Materials and Methods. Results are presented as means ± SD. An unequal variance Welch t test was performed (**P value of 0.0079). (C) ARAF H-scores of the tumor samples are illustrated dependent on the TMN-N classification of the corresponding patients. N0 indicates no signs of lymph node (LN) involvement, and patients with N1 to N3 have affected lymph nodes. ARAF stainings with H-scores < 50 are classified as ARAF negative, and ARAF stainings with H-scores > 50 are classified as ARAF-positive samples.
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