Hyperactive KRAS/MAPK signaling disrupts normal lymphatic vessel architecture and function

Abstract

Complex lymphatic anomalies (CLAs) are sporadically occurring diseases caused by the maldevelopment of lymphatic vessels. We and others recently reported that somatic activating mutations in KRAS can cause CLAs. However, the mechanisms by which activating KRAS mutations cause CLAs are poorly understood. Here, we show that KRAS^G12D expression in lymphatic endothelial cells (LECs) during embryonic development impairs the formation of lymphovenous valves and causes the enlargement of lymphatic vessels. We demonstrate that KRAS^G12D expression in primary human LECs induces cell spindling, proliferation, and migration. It also increases AKT and ERK1/2 phosphorylation and decreases the expression of genes that regulate the maturation of lymphatic vessels. We show that MEK1/2 inhibition with the FDA-approved drug trametinib suppresses KRAS^G12D-induced morphological changes, proliferation, and migration. Trametinib also decreases ERK1/2 phosphorylation and increases the expression of genes that regulate the maturation of lymphatic vessels. We also show that trametinib and Cre-mediated expression of a dominant-negative form of MEK1 (Map2k1 ^K97M) suppresses KRAS^G12D-induced lymphatic vessel hyperplasia in embryos. Last, we demonstrate that conditional knockout of wild-type Kras in LECs does not affect the formation or function of lymphatic vessels. Together, our data indicate that KRAS/MAPK signaling must be tightly regulated during embryonic development for the proper development of lymphatic vessels and further support the testing of MEK1/2 inhibitors for treating CLAs.

Keywords: Gorham-Stout disease; KRAS; complex lymphatic anomaly; lymphangiogenesis; lymphatic malformation; trametinib.

FIGURE 1

LEC ^KrasG12D embryos have enlarged jugular lymph sacs and malformed lymphovenous valves. (A). Schematics of the Lyve1-Cre and Kras ^LSL-G12D alleles. (B). Representative images of E14.5 LEC ^Ctrl and LEC ^KrasG12D embryos. The LEC ^KrasG12D embryo has edema. (C). Transverse sections of E14.5 embryos stained with an anti-Lyve1 antibody (brown) and hematoxylin (purple). (D). Jugular lymph sac area was significantly greater in LEC ^KrasG12D embryos (273391 ± 47694; n = 6 mice) than LEC ^Ctrl embryos (76949 ± 17566; n = 5 mice). (E). Coronal sections of E14.5 embryos stained with hematoxylin and eosin (H&E). The arrow points to a lymphovenous valve in a LEC ^Ctrl embryo. The arrow points to a cluster of cells in the lymphovenous valve region in a LEC ^KrasG12D embryo. Six LEC ^Ctrl and five LEC ^KrasG12D embryos were analyzed. (F). Coronal sections of E14.5 embryos stained with DAPI (blue) and antibodies against Lyve1 (yellow) and endomucin (red). The arrow points to a lymphovenous valve in a LEC ^Ctrl embryo. The arrow points to a cluster of Lyve1-positive cells in the lymphovenous valve region in a LEC ^KrasG12D embryo. Five LEC ^Ctrl and three LEC ^KrasG12D embryos were analyzed. (G). Immunostaining revealed that cell clusters in the jugular lymph sacs of LEC ^KrasG12D embryos contained Prox1 and Lyve-1 double-positive LECs. Six LEC ^KrasG12D embryos were analyzed. (H). Higher magnification image of cluster in panel (G). (I,J). Clusters also contained F4/80-positive cells (I) and CD45-positive cells (J). (K). Immunostaining revealed that clusters in LEC ^KrasG12D embryos contained Runx1-positive cells. (L–O). Higher magnification images of the separate channels in panel (K). Data are presented as mean ± SEM. **p < 0.01; unpaired Student’s t-tests. Scale bar in panel C = 200 µm. Scale bars in panels E, G, I, J, and K = 100 µm.

FIGURE 2

LEC ^KrasG12D embryos have abnormal dermal lymphatic vessels. (A,B). Back skin whole-mounts from E15.5 embryos stained for neuropilin-2. (C). LEC ^KrasG12D embryos (11.00 ± 1.732; n = 3 mice) had significantly fewer lymphatic vessel branch points than LEC ^Ctrl embryos (62.14 ± 8.382; n = 7 mice). (D). Lymphatic vessel diameter was significantly greater in LEC ^KrasG12D embryos (151.4 ± 20.05; n = 3 mice) than in LEC ^Ctrl embryos (43.30 ± 2.047; n = 7 mice). Data are presented as mean ± SEM. ** p < 0.01, **** p < 0.0001; unpaired Student’s t-tests. Scale bar = 250 µm.

FIGURE 3

KRAS^G12D induces cell morphological changes, proliferation, and migration. (A). Brightfield images of GFP-LECs and KRAS^G12D-LECs. The images were taken 72 h after treating primary human LECs with lentivirus particles that express GFP or KRAS^G12D. GFP-LECs exhibit a normal cobblestone morphology, whereas KRAS^G12D-LECs exhibit a spindle morphology. (B). Circularity index measurements for GFP-LECs and KRAS^G12D-LECs. (C). MTS viability assay results for GFP-LECs and KRAS^G12D-LECs. Viability was measured 72 h after treating cells with lentivirus particles that express GFP or KRAS^G12D. (D). Representative images of GFP-LECs and KRAS^G12D-LECs taken 0 and 14 h after scratching confluent monolayers of cells. (E). Graph showing scratch closure area 14 h after wounding. KRAS^G12D-LECs closed the scratched area significantly faster than GFP-LECs. Data are presented as mean ± SEM. **p < 0.01, ****p < 0.0001; unpaired Student’s t-tests. Scale bars = 300 µm.

FIGURE 4

KRAS^G12D expression in LECs induces PI3K and MAPK signaling and changes in gene expression. (A). Western blot results for phospho-AKT, AKT, phospho-ERK1/2, ERK1/2, GFP, KRAS^G12D (mutation-specific antibody), and tubulin. Protein lysates were made 72 h after treating primary human LECs with GFP or KRAS^G12D expressing lentivirus particles. (B). Graphs of Western blot results. Phospho-AKT and phospho-ERK1/2 levels were significantly higher in KRAS^G12D-LECs than GFP-LECs. GFP-LECs specifically expressed GFP, and KRAS^G12D-LECs specifically expressed KRAS^G12D. (C). Volcano plot of RNA-Seq results comparing KRAS^G12D-LECs to GFP-LECs. Two thousand and nine genes were upregulated (red dots), and 1,396 genes were downregulated (blue dots) by KRAS^G12D (log₂ fold-change ≥1 or ≤ −1; FDR <0.01). D,E. Select GO terms associated with genes upregulated by KRAS^G12D (D) and genes downregulated by KRAS^G12D (E). (F,G). Heatmaps of genes that regulate angio/lymphangiogenesis (F) and genes that control lymphatic valve development or lymphatic muscle cell recruitment (G). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01; unpaired Student’s t-tests.

FIGURE 5

Trametinib decreases KRAS^G12D-induced cell shape changes, proliferation, and migration. (A). Representative brightfield images of GFP-LECs and KRAS^G12D-LECs treated with DMSO or trametinib (10 nM) for 48 h. (B). Circularity index measurements for GFP-LECs and KRAS^G12D-LECs treated with DMSO or trametinib (10 nM). Trametinib significantly increased the circularity of KRAS^G12D-LECs. (C). MTS viability assay results for GFP-LECs and KRAS^G12D-LECs treated with DMSO or trametinib (10 nM) for 72 h. Trametinib decreased the proliferation of KRAS^G12D-LECs. (D). Representative images of GFP-LECs and KRAS^G12D-LECs taken 0 or 14 h after scratching confluent monolayers of cells. Cells were treated with DMSO or trametinib (10 nM) immediately after scratching. (E). Graph showing scratch closure area 14 h after wounding. Trametinib-treated KRAS^G12D-LECs closed the scratched area significantly slower than DMSO-treated KRAS^G12D-LECs. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001, ns = not significant; ANOVA Tukey’s multiple comparisons test. Scale bars = 300 µm.

FIGURE 6

Trametinib decreases MAPK signaling and increases the expression of lymphatic maturation genes. (A). Western blot analysis of phospho-AKT, AKT, phospho-ERK1/2, ERK1/2, GFP, KRAS^G12D (mutation-specific antibody), and tubulin. Protein lysates were made 16 h after treating GFP-LECs and KRAS^G12D-LECs with DMSO or trametinib (10 nM). (B). Graphs of Western blot results. Trametinib increased phospho-AKT levels and decreased phospho-ERK1/2 levels in KRAS^G12D-LECs. (C). Volcano plot of RNA-Seq data comparing trametinib-treated KRAS^G12D-LECs to DMSO-treated KRAS^G12D-LECs. RNA was isolated 16 h after treating cells with DMSO or trametinib (10 nM). Eight hundred forty-one genes were upregulated (red dots), and 424 genes were downregulated (blue dots) by trametinib (log₂ fold-change ≥1 or ≤ −1; FDR <0.02). (D,E). Select GO terms associated with genes upregulated by trametinib (D) and genes downregulated by trametinib (E). (F,G). Heatmaps of genes that regulate angio/lymphangiogenesis (F) and genes that control lymphatic valve development or lymphatic muscle cell recruitment (G). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001, ns = not significant; ANOVA Tukey’s multiple comparisons test and Dunnett’s multiple comparisons test.

FIGURE 7

Trametinib partially suppresses KrasG12D-induced enlargement of lymphatic vessels. (A). Schematic showing when mice received vehicle or trametinib (1 mg/mL; p.o.; q.d.). (B). Lymphatic vessel branch points for vehicle-treated LEC ^Ctrl (90.50 ± 3.647; n = 5 mice), trametinib-treated LEC ^Ctrl (93.30 ± 9.332; n = 5 mice), vehicle-treated LEC ^KrasG12D (25.97 ± 3.578; n = 5 mice), and trametinib-treated LEC ^KrasG12D mice (23.50 ± 4.363; n = 6 mice). (C). Lymphatic vessel diameter measurements for vehicle-treated LEC ^Ctrl (46.24 ± 1.872; n = 5 mice), trametinib-treated LEC ^Ctrl (56.02 ± 4.381; n = 5 mice), vehicle-treated LEC ^KrasG12D (152.4 ± 14.14; n = 5 mice), and trametinib-treated LEC ^KrasG12D mice (85.42 ± 7.590; n = 6 mice). (D). Back skin whole-mounts from E14.5 embryos stained for neuropilin-2. Data are presented as mean ± SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001, ns = not significant; ANOVA Tukey’s multiple comparisons test. Scale bar = 250 µm.

FIGURE 8

A dominant-negative form of MEK1 (Map2k1^K97M) partially suppresses Kras^G12D-induced enlargement of lymphatic vessels. (A). Schematics of the Lyve1-Cre, Kras ^LSL-G12D , and Tg ^{LSL-Map2k1K97M} alleles. (B). Lymphatic vessel branch points for LEC ^Ctrl (90.38 ± 9.783; n = 10 mice), LEC ^Map2k1K97M (101.8 ± 5.693; n = 13 mice), LEC ^KrasG12D (18.38 ± 2.151; n = 7 mice), and LEC ^{Map2k1K97M;KrasG12D} (32.33 ± 6.320; n = 4 mice). (C). Lymphatic vessel diameter measurements for LEC ^Ctrl (47.54 ± 2.628; n = 10 mice), LEC ^Map2k1K97M (37.05 ± 1.972; n = 13 mice), LEC ^KrasG12D (189.2 ± 6.192; n = 7 mice), and LEC ^{Map2k1K97M;KrasG12D} (100.4 ± 6.485; n = 4 mice). The Tg ^{LSL-Map2k1K97M} allele significantly decreased lymphatic vessel diameter in LEC ^KrasG12D embryos. (D). Back skin whole-mounts from E15.5 embryos stained for neuropilin-2. Data are presented as mean ± SEM. **** p < 0.0001, ns = not significant; ANOVA Tukey’s multiple comparisons test. Scale bar = 250 µm.

FIGURE 9

Conditional knockout of Kras in LECs does not impair the development or function of lymphatic vessels. (A). Schematics of the Lyve1-Cre and Kras ^loxp alleles. (B). RT-PCR results for Kras and Gapdh using cDNA generated from RNA isolated from pulmonary endothelial cells from LEC ^Ctrl and LEC ^ΔKras mice. (C). Ear skin whole-mount preparations from LEC ^Ctrl and LEC ^ΔKras mice stained for Lyve1. (D). The number of lymphatic vessel branch points was not significantly different between LEC ^Ctrl (21.68 ± 1.049; n = 6 mice) and LEC ^ΔKras mice (24.10 ± 0.9766; n = 5 mice). (E). The diameter of lymphatic vessels was not significantly different between LEC ^Ctrl (43.05 ± 1.669; n = 6 mice) and LEC ^ΔKras mice (47.47 ± 1.141; n = 5 mice). (F). Ear skin whole-mount preparations from LEC ^Ctrl and LEC ^ΔKras mice stained for Lyve1, CD31, and Vegfr3. (G). The number of lymphatic valves/mm vessel was not significantly different between LEC ^Ctrl (0.4812 ± 0.06282; n = 5 mice) and LEC ^ΔKras mice (0.5495 ± 0.02791; n = 6 mice). (H). Intradermally injected Evans blue dye was effectively transported from injection sites in LEC ^Ctrl (n = 4 mice) and LEC ^ΔKras mice (n = 4 mice). Data are presented as mean ± SEM. ns = not significant; unpaired Student’s t-tests. Scale bar in panel C = 250 µm. Scale bar in panel F = 100 µm.