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. 2019 Aug 27;8:e43668. doi: 10.7554/eLife.43668

Figure 5. BRAFV600E/PI3KαH1047R and BRAFV600E driven tumors both show effects on differentiation status, with BRAFV600E/PI3KαH1047R driven tumors displaying more profound effects on identity.

(A) BRAFV600E/PI3KαH1047R and BRAFV600E driven hyperplasia both show immunoreactivity of the AT1 marker, AQP5, 2 weeks post initiation with BRAFV600E/PI3KαH1047R driven hyperplasia showing enhanced immunostaining. BRAFV600E/PI3KαH1047R and BRAFV600E driven tumors also show immunoreactivity of AQP5 12 weeks post initiation with BRAFV600E/PI3KαH1047R driven tumors showing a more variable pattern of immunostaining. Scale bars = 100 um. (B) Quantitation demonstrating significant effect of PI3KαH1047R on AQP5 immunoreactivity in BRAFV600E driven tumors 2 weeks post initiation. No difference seen in AQP5 immunoreactivity between BRAFV600E/PI3KαH1047R and BRAFV600E driven tumors 12 weeks post initiation. This appears to be the result of a slight increase in AQP5 immunoreactivity between 2 and 12 weeks in BRAFV600E driven tumors and a more dramatic decrease in AQP5 immunoreactivity between 2 and 12 weeks in BRAFV600E/PI3KαH1047R driven tumors. ANOVA p<1e-5, multiple comparisons done by Tukey’s Honest Significant Difference test, ****: p<1 e −5, **p=0.0014. (C) BRAFV600E driven tumors display widespread immunoreactivity to both AQP5 and the AT2 marker, LYZ, 12 weeks post initiation. BRAFV600E/PI3KαH1047R driven tumors show cells with widely varied expression of differentiation markers, including AQP5+, LYZ+ (Cyan arrows); AQP5-, LYZ+ (Red arrows); AQP5+, LYZ- (Green arrows); and AQP5-, LYZ- (Yellow arrows) cells. Scale bars = 100 um. (D) BRAFV600E driven tumors show relatively high association between AQP5 and LYZ immunoreactivity (Rho = 0.54). (E) BRAFV600E/PI3KαH1047R driven tumors show relatively low association between AQP5 and LYZ immunoreactivity (Rho = 0.13) (F) Overlay of BRAFV600E/PI3KαH1047R and BRAFV600E driven tumors 12 weeks post initiation. Dashed line for each marker drawn at mean - one standard deviation of BRAFV600E driven tumors. BRAFV600E/PI3KαH1047R driven tumors show fewer AQP5+, LYZ + cells, most strongly accounted for by an increase in AQP5+, LYZ- cells, but with increases also seen in AQP5-, LYZ + and AQP5-, LYZ- cells. Chi square test associates genotype with distribution, p val <1e-5.

Figure 5—source code 1. R script to perform statistics on Figure 4—source data 1, as well as plot these results.
elife-43668-fig5-code1.r (7.6KB, r)
DOI: 10.7554/eLife.43668.027
Figure 5—source code 2. Cellprofiler pipeline to quantify raw images from BRAFV600E/PI3KαH1047R and BRAFV600E driven tumors, producing Figure 5—source data 1.
elife-43668-fig5-code2.cpproj (1.3MB, cpproj)
DOI: 10.7554/eLife.43668.028
Figure 5—source data 1. Cellprofiler output quantifying AQP5 and LYZ immunofluorescence in BRAFV600E/PI3KαH1047R and BRAFV600E driven tumors.
elife-43668-fig5-data1.zip (41.8MB, zip)
DOI: 10.7554/eLife.43668.029

Figure 5.

Figure 5—figure supplement 1. BRAFV600E-driven lung tumors show characteristics of bipotent progenitor cells.

Figure 5—figure supplement 1.

(A) Electron micrograph showing a normal AT2 cell in the lung epithelium. Cyan structures are surfactant rich lamellar bodies, red structure is nucleus, green structures are mitochondria. Scale bar = 2 um. (B) Electron micrograph showing an abnormal AT2 cell in a BRAFV600E driven tumor harboring a large vacuole (purple). Scale bar = 2 um. (C) Enhanced magnification of vacuole showing rough electron dense pattern characteristic of glycogen storage. Scale bar = 500 nm. (D) Human lung adenocarcinomas harboring mutations in either PIK3CA, PTEN, or AKT show a significant reduction in PPARGC1A transcript levels when compared to tumors not shown to be harboring any of these three mutations (p=0.0295 by unpaired t-test with Welch’s correction). (E) 5 kb promoter regions of AT2 specific genes show enrichment of novel DNA motifs. The most significantly enriched of these motifs (E,1) is represented in 71 of 99 promoters scanned and significantly matches the known motif of NR5A2. (F) Alignment of the consensus binding motif for NR5A2 with the novel conserved motifs found in E,1 and E,4.