Loss of <i>DOK2</i> in human lung adenocarcinoma is associated with <i>EGFR</i> mutation.

<p>(<b>A</b>) Association between <i>EGFR</i> or <i>KRAS</i> mutation status and genomic loss of the <i>DOK2</i> locus from aCGH analysis of 199 primary human lung adenocarcinomas [20]. ****, <i>P</i> < 0.001. *, P < 0.05 by Fisher’s exact test. aCGH analysis and mutation calling of tumors was determined as previously described [20]. (<b>B</b>) Quantitative representation of data shown in (A). Data shown is mean+SEM of log₂ ratio data from array CGH data. ***, <i>P</i> < 0.001. *, <i>P</i> < 0.05 by two-tailed unpaired t-test. (<b>C</b>) Copy number and mutation associations in The Cancer Genome Atlas (TCGA) data from 230 lung adenocarcinomas. Copy number and mutation data were downloaded from TCGA (<a href="https://tcga-data.nci.nih.gov/" target="_blank">https://tcga-data.nci.nih.gov/</a>). </p

<i>DOK2</i> fails to inhibit tumor formation of <i>KRAS</i>-mutant lung adenocarcinoma cells.

<p>(<b>A</b>) Tumor volume of A549 cells after xenografting into nude mice. A549 cells were transduced with retrovirus to generate stable cell lines with or without expression of DOK2 and then each line was injected subcutaneously into the flanks of nude mice. Data shown are mean +/- SD of three replicates. (<b>B</b>) RAS activity in A549 cells with or without DOK2 expression. The cells were serum starved and then stimulated with 100 ng/ml EGF for the indicated time, then lysed. GTP-bound RAS was isolated from lysates via a RAF-binding domain (RBD) pulldown, and the pulldown fraction (top panel) and total lysate (bottom panels) were analyzed by Western blotting using anti-RAS, DOK2, or HSP90 (loading control) antibodies. </p

<i>Dok2</i> suppresses lung tumorigenesis initiated by oncogenic <i>EGFR.</i>

<div><p>(<b>A</b>) MR images from the lungs of <i>C/EGFR^DEL/Dok2</i>^<i>+/+</i> and <i>C/EGFR^DEL/Dok2</i>^<i>-/-</i> mice after 12 months of doxycycline treatment. Images from four individual animals of each genotype are shown. Arrowheads indicate tumor nodules. h, heart. Signal not indicated by arrows or arrowheads is likely to be diffuse hyperplasia or bronchoalveolar carcinoma (BAC). (<b>B</b>) H&E staining of lungs from <i>C/EGFR^DEL/Dok2</i>^<i>+/+</i> and <i>C/EGFR^DEL/Dok2</i>^-/- mice after 12 months of doxycycline treatment. 20X total magnification. For each genotype, four lung lobes from a single representative mouse are shown.</p> <p>(<b>C</b>) Lung weight of lungs from <i>C/EGFR^DEL/Dok2</i>^<i>+/+</i> and <i>C/EGFR^DEL/Dok2</i>^<i>-/-</i> mice after 12 months of doxycycline treatment. Data shown is mean + SEM. *, <i>P</i> < 0.05 by two-tailed t-test. <i>C</i>/EGFR^DEL/Dok2^+/+, n = 8. <i>C/EGFRDEL/Dok2</i>^<i>-/-</i>, n = 5. (<b>D</b>) Tumors per slide per animal in <i>C/EGFR^DEL/Dok2</i>^<i>+/+</i>, <i>C/EGFR^DEL/Dok2</i>^<i>-/-</i>, and age-matched non-transgenic <i>Dok2</i> KO mice. Data shown is mean + SEM. **, <i>P</i> < 0.01 by two-tailed t-test. <i>C</i>/EGFR^DEL/Dok2^+/+, n = 5. <i>C/EGFRDEL/Dok2</i>^<i>-/-</i>, n = 4. Non-transgenic <i>Dok2</i> KO, n = 4. (<b>E</b>) Kaplan-Meier plot of survival data from bitransgenic <i>C</i>/EGFR^DEL/Dok2^+/+ (n = 29), <i>C</i>/EGFR^DEL/Dok2^-/- (n = 39), and non- or mono-transgenic littermate controls of all <i>Dok2</i> genotypes (n = 71). Spontaneous deaths or sacrifices due to poor body condition were recorded as events. Planned sacrifices at other time points were censored.</p></div

EGFR activity regulates binding of DOK2 to EGFR and DOK2 localization.

<p>(<b>A</b>) Co-immunoprecipitation of DOK2, EGFR, and RASA1. HEK293 cells were co-transfected with pCMV-DOK2 (all conditions) and either pcDNA3.1 empty vector or pcDNA3.1-EGFR^WT, pcDNA3.1-EGFR^L858R, or pcDNA3.1-HA-HRAS^G12V. Cells were serum starved overnight in medium containing 0.1% FBS before stimulation with 50 ng/ml EGF for 2-5 minutes. DOK2 and associated proteins were immunoprecipitated from extracts of stimulated and unstimulated cells with an anti-DOK2 antibody and then analyzed by Western blotting. Upper panels show the proteins in the immunoprecipitation fraction; lower panels show proteins from the total lysate. A black arrowhead indicates HA-HRAS^G12V whereas a white arrowhead indicates endogenous RAS. Data shown is representative of results from at least three independent experiments. (<b>B</b>) Immunofluorescence of NIH3T3 cells stably expressing WT or EGFR^L858R. Cells were transfected with pCMV-DOK2, incubated overnight, serum starved, and then either fixed or stimulated with EGF before fixation. The left panels show DOK2 (green) and DAPI (blue). White arrowheads indicate membrane-associated DOK2 staining. A Western blot indicates total levels of EGFR in these cells (right panel). Data shown is representative from at least three independent experiments. (<b>C</b>) Confocal microscopy analysis of colocalization of DOK2 and EGFR. 3T3-EGFR^L858R cells were transfected with FLAG-DOK2 and probed with anti-FLAG (red) or anti-EGFR (green) antibodies. Arrowheads indicate colocalization (yellow) in the panel showing the merged signals. Data shown is representative from at least three independent experiments.</p

<i>DOK2</i> inhibits tumor formation of <i>EGFR</i>-mutant lung adenocarcinoma cells.

<p>(<b>A</b>) Tumor volume of NCI-H1975 cells after xenografting into nude mice. NCI-H1975 cells were transduced with retrovirus to generate stable cell lines with or without expression of DOK2 and each line was then injected subcutaneously into the flanks of nude mice. Data shown are mean +/- SD of three replicates. (<b>B</b>) Weight of tumors formed by NCI-H1975 cells with or without expression of DOK2. Pictures (inset) were taken at 8 weeks post-injection. (<b>C</b>) RAS activity in NCI-H1975 cells with or without DOK2 expression. The cells were serum starved and then stimulated with 100 ng/ml EGF for the indicated time, then lysed. GTP-bound RAS was isolated from lysates via a RAF-binding domain (RBD) pulldown, and the pulldown fraction (top panel) or total lysate (bottom panels) were analyzed by Western blotting using anti-RAS, DOK2, or HSP90 (loading control) antibodies. </p

Loss of <i>Dok2</i> fails to impact <i>Kras</i>-mutant lung tumorigenesis.

<p>(<b>A</b>) MR images of the lungs of <i>C/Kras^G12D/Dok2</i>^<i>+/+</i> and <i>C/Kras^G12D/Dok2</i>^-/- lungs after 5 months of doxycycline induction. h, heart. L, liver. (<b>B</b>) H&E staining of lungs from <i>C/Kras^G12D/Dok2</i>^<i>+/+</i> and <i>C/Kras^G12D/Dok2</i>^-/- after 5 months of doxycycline treatment. 20X total magnification. For each genotype, four lung lobes from a single representative mouse are shown. (<b>C</b>) Lung weight data from 4-5 month old animals. Mean + SEM is shown from n = 6 control (non-transgenic) mice and n = 4 <i>C/Kras^G12D/Dok2</i>^<i>+/+</i> and <i>C/Kras^G12D/Dok2</i>^-/- animals. (<b>D</b>) Kaplan-Meier curve showing survival data from <i>C/Kras^G12D/Dok2</i>^<i>+/+</i> and <i>C/Kras^G12D/Dok2</i>^-/- and non- or mono-transgenic controls (<i>Dok2</i> +/+ and -/-) treated with doxycycline for the indicated times. </p