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> 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

<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

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

Pten Dose Affects Prostate Tumor Progression

<p><i>Pten</i>^+/− mice develop hyperplasia, dysplasia, and low-grade PIN. <i>Pten</i>^hy/− mice develop at complete penetrance high-grade PIN at a young age (8–10 wk) and roughly 25% present invasive CaP around 8 mo. <i>Pten</i>^pc mice develop invasive CaP at complete penetrance. (See Discussion for a detailed description.) <i>p27^Kip1−/−</i> mice, on the contrary, develop only BPH. Possibilities for human therapeutic intervention derived from our findings: in addition to inactivation of PI3K/AKT and mTOR enzymatic activities (in <i>PTEN</i> loss of heterozygosity condition), monitoring and elevation of PTEN expression levels of the remaining allele could not only prevent formation of PIN lesions (in <i>PTEN</i>^+/− individuals), but could importantly also be used to counteract the progression to invasive phenotypes (as observed in <i>Pten</i>^hy/−<i></i>mouse mutants).</p

Production and Analysis of a <i>Pten</i> Hypomorphic Mouse Series

<div><p>(A)<b> </b> Schematic representation of the wild-type (<i>+</i>), hypomorphic (<i>hy</i>), and null (−) alleles of <i>Pten</i>. (For a detailed view, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000059#pbio.0000059-g003" target="_blank">Figure 3</a>A.)</p> <p>(B) Breeding scheme used to produce a <i>Pten</i> hypomorphic mouse series and predicted hierarchy of <i>Pten</i> expression levels.</p> <p>(C) WB analysis of MEF lysates from ten littermate primary cell cultures obtained from a single cross (indicated in [B]) confirms predicted <i>Pten</i> expression hierarchy in the hypomorphic series and inversely related Akt phosphorylation status (top), both verified by densitometric analysis and plotting of the Pten/actin and phospho-Akt/Akt ratios (bottom).</p> <p>(D) MEF cDNA was amplified by semiquantitative PCR with exon 3 (forward) and exon 4–5 spanning (reverse) primers for <i>Pten</i>. Consistent with the concept of transcriptional interference at the <i>Pten</i> locus, <i>Pten</i> mRNA levels in <i>Pten</i>^hy/− MEFs are clearly below the level observed in heterozygosity. Lower panels show the quantification of <i>Pten</i> relative to <i>Hprt</i> amplification.</p></div

Molecular Effects of Loss of <i>Pten</i> and Biological Comparison of All Generated Mouse Models

<div><p>(A) Ki-67 staining of AP lobe sections illustrates increasing proliferation with decreasing Pten levels (numbers are Ki-67-positive cells per 300 cells counted in percent; bars are 50 μm).</p> <p>(B) The phospho-Akt/Akt ratio is sharply increased in the prostates of 10-wk-old <i>Pten</i>^pc2 animals, as shown by densitometric quantification of WB analysis.</p> <p>(C) AP staining with anti-phospho-Akt antibodies reveals strong plasma membrane localization of phospho-Akt and apparent reduction of p27 protein detection, whereas phospho-mTOR and phospho-threonine-FOXO3 antibodies show increased staining in <i>Pten</i>^pc2 versus wild-type mouse prostates. Bars are 50 μm.</p> <p>(D) Kaplan–Meier curve showing prostate enlargement as visualized by MRI. Progressive rates of mass increase for <i>Pten</i>^pc2 (median age, 4 mo), <i>Pten</i>^hy/− (median age, 7 mo), and <i>Pten</i>^pc1 (median age, 16 mo) mice are found. In contrast, no prostatic size irregularities were detected in <i>Pten^+/+</i> or <i>Pten^+/−</i> mice.</p> <p>(E) Incidence of invasive CaP. Invasive CaP was defined as tumor cells disrupting the basal membrane of prostatic glands and growing into the surrounding stroma. Full penetrance was observed in both <i>Pten^pc1</i> mice as well as in <i>Pten^pc2</i> mice. In contrast, <i>Pten^hy/−</i> mice with a follow-up of more than 6 mo displayed only 25% incidence of invasive CaP.</p></div