1. Volume 18, Issue 8, Pages 1958-1969 (February 2017)
Context-Dependent Effects of Amplified MAPK Signaling during Lung Adenocarcinoma Initiation and Progression 
Michelle Cicchini, Elizabeth L. Buza, Kyra M. Sagal, A. Andrea Gudiel, Amy C. Durham, David M. Feldser 
Cell Reports 
Volume 18, Issue 8, Pages (February 2017)
DOI: /j.celrep
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2. Cell Reports 2017 18, 1958-1969DOI: (10.1016/j.celrep.2017.01.069)
Copyright © 2017 The Authors Terms and Conditions

3. Figure 1
MAPK Signal Amplification Promotes Early and Widespread Lung Tumor Growth in KrasLSL-G12D/+;p53flox/flox Mice
(A) KrasLSL-G12D/+;p53flox/flox mice were infected with Ad:CMVCre by forced inhalation to initiate lung tumors. One week post-Cre, treatment with control or PLX4720-containing diet was initiated and maintained for the indicated durations.
(B) Kaplan-Meier survival analysis of Ad:CMVCre-infected mice that were maintained on control or PLX4720-containing diets; p < ; log rank (Mantel-Cox) test.
(C) H&E images of lung tumors and hyperplasias from mice at the survival endpoint.
(D) Tumor grades in mice treated with control or PLX4720 for survival analysis (contingency χ2 test for trend; p < ; Student’s t test of mean number of AAH per mouse; p = 0.08).
(E) Immunohistochemistry for pMek and pErk 5 weeks post-Ad:CMVCre.
(F and G) KrasLSL-G12D/+;p53flox/flox mice were infected with 5 × 107 or 5 × 106 PFUs of Ad:CMVCre as indicated. (F) Percent lung tumor burden after 5 weeks after Ad:CMVCre infection is shown; p < (G) Tumor number in lungs of mice 5 weeks after Ad:CMVCre infection is shown; p =
(H) Whole-lung lobes (upper panel) and lung sections (middle and lower panels) analyzed by fluorescence microscopy from KrasLSL-G12D/+; p53flox/flox; Rosa26Motley/+ mice 5 weeks after Ad:CMV-Cre infection.
The scale bars represent 25 μm. Error bars represent SEM from average. Also see Figures S1 and S2.
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4. Figure 2
Enhancement of Tumor Growth from Pharmacological MAPK Signal Amplification Is Mek1/2 Dependent
(A) KrasLSL-G12D/+;p53flox/flox mice were infected with 5 × 107 PFUs of Ad:CMVCre to initiate tumor formation. One week post-infection, control, PLX4720 (Braf inhibitor), or PLX4720/PD (Braf inhibitor/Mek1/2 inhibitor) treatments were maintained for 4 weeks.
(B) H&E images of whole lung lobes (upper panel) and high magnification images (lower panel) from areas showing hyperplasias.
(C and D) Percent lung tumor burden (C) and tumor number (D) analysis 5 weeks after Ad:CMVCre infection.
(E) Immunohistochemistry for pMek and pErk in mice 5 weeks post-Ad:CMVCre.
(F) pErk, total Erk (tErk), and Hsp90 expression in whole-lung lysates of normal, control, PLX4720-, or PLX4720/PD treated mice; t = 1–5 weeks.
The scale bars represent 25 μm. Error bars represent SEM from average. Also see Figure S3.
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5. Figure 3
MAPK Amplification Drives Cell Proliferation and Tumor Progression in Established p53-Deficient Tumors
(A) KrasG12D/+;p53flox/flox mice are infected with Ad:CMVCre to initiate tumor formation. Starting 4 or 7 weeks post-Cre, mice were treated with either control or PLX4720 until 9 weeks.
(B) H&E, pMek, pErk, and Arf staining of lung tumors from mice on control or PLX4720; t = 7–9 weeks.
(C) Western blot analysis of lysates from individually isolated tumors for pErk, tErk, and Hsp90; mice treated t = 4–9 weeks.
(D) BrdU and Nkx2-1 lung tumor staining from control or PLX4720-treated mice. Mice were injected with BrdU at days 6 and 13 after starting control or PLX4720 treatments.
(E) Quantification of BrdU and Nkx2-1 dual positive cells from mice on control or PLX4720 treatments from t = 7–9 weeks; p <
(F and G) Percent lung tumor burden (F), p = 0.026, and tumor grades (G) in mice treated from t = 7–9 weeks with control or PLX4720 (contingency χ2 test for trend; p = ).
The scale bars represent 100 μm. Error bars represent SEM from average. Also see Figure S4.
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6. Figure 4
MAPK Signal Amplification Leads to Massive Expansion of Low-Grade Tumors despite Wild-Type p53
(A) KrasLSL-G12D/+;p53+/+ mice infected with Ad:CMV-Cre were treated 1 week later with control or PLX4720.
(B) H&E images of whole lung lobes 8 weeks after Ad:CMV-Cre infection.
(C) Percent lung tumor burden analysis in mice 8 weeks after Ad:CMV-Cre infection.
(D) Quantification of phosphorylated-histone 3 (p-H3)-positive cells.
(E) H&E and immunostaining for pMek and pErk of lung sections 5 weeks after Ad:CMV-Cre infection.
(F) pErk, tErk, and Hsp90 expression from whole-lung lysates of KrasLSL-G12D/+ and KrasLSL-G12D/+;p53flox/flox mice treated with control or PLX4720 from t = 1–5 weeks.
(G) Quantification of MAPK signaling in lysates from whole lung lobes of KrasLSL-G12D/+;p53+/+ and KrasLSL-G12D/+;p53flox/flox mice expressed as a ratio of normalized pErk to tErk for each sample.
(H) Tumor grades in KrasLSL-G12D/+;p53+/+ mice 8 weeks after Ad:CMV-Cre infection. Cumulative tumor number score is indicated. Average number of lesions per mouse was significantly higher in the PLX4720 group (p = 0.02), but tumor grade distribution was not different (contingency χ2 test for trend; p = ).
The scale bars represent 100 μm. Error bars represent SEM from average. Also see Figure S5.
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7. Figure 5
MAPK Signal Amplification Promotes Tumor Initiation in AT2 and Club Cells
(A) KrasG12D/+;p53flox/flox;Rosa26LSL-YFP/+ mice were infected with Ad:SPC-Cre or Ad:CC10-Cre to initiate tumor formation. One week post-infection, control or PLX4720 treatments began, and lung tissue was analyzed 4 weeks later.
(B) Immunohistochemistry of serial lung sections for YFP and pErk. The scale bars represent 50 μm.
(C) Immunofluorescence for YFP (yellow). The scale bars represent 100 μm.
(D) Contingency tables of YFP-positive singlet cells from (C). Significance is indicated; Fisher’s exact test.
Also see Figure S6.
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8. Figure 6
MAPK Signal Amplification Drives Transdifferentiation of Club Cells toward AT2 Cell Fate
Experimental scheme is identical to Figure 5A.
(A) Immunostaining of lung sections from KrasLSL-G12D/+;p53flox/flox;Rosa26LSL-YFP/+ mice. DAPI (white), YFP (yellow), CC10 (cyan), and SPC (red).
(B) The pink and green boxes highlight regions from (A), for PLX4720 treated Ad:CC10-Cre mice. The pink box indicates SPC-positive adenoma adjacent to CC10-positive club cells in the airway. The green box highlights hyper-proliferation in the airway positive for SPC and CC10.
(C) Immunostaining for YFP (yellow), Sox2 (cyan), and DAPI (white) in the airways and adenomas. Hyperplasia in the airway demonstrates YFP-positive cells that have lost Sox2 expression (white arrows). The pink arrow highlights YFP-positive neoplasia retaining Sox2 expression along airway transitioning to Sox2-negative adenomas in the alveolar space.
(D) Quantification of YFP+ lesions based on Sox2 expression status; Fisher’s exact test; p < The scale bars represent 100 μm.
(E) Model: MAPK signal amplification alters tumor initiation and progression in lung epithelial cell types. See text for details.
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