P-Rex1 is required for efficient melanoblast migration and melanoma metastasis

Abstract

Metastases are the major cause of death from melanoma, a skin cancer that has the fastest rising incidence of any malignancy in the Western world. Molecular pathways that drive melanoblast migration in development are believed to underpin the movement and ultimately the metastasis of melanoma. Here we show that mice lacking P-Rex1, a Rac-specific Rho GTPase guanine nucleotide exchange factor, have a melanoblast migration defect during development evidenced by a white belly. Moreover, these P-Rex1(-/-) mice are resistant to metastasis when crossed to a murine model of melanoma. Mechanistically, this is associated with P-Rex1 driving invasion in a Rac-dependent manner. P-Rex1 is elevated in the majority of human melanoma cell lines and tumour tissue. We conclude that P-Rex1 has an important role in melanoblast migration and cancer progression to metastasis in mice and humans.

Figure 1

P-Rex1-deficient mice have a ‘white belly’ phenotype (a) Belly and feet of P-Rex1^+/+ and P-Rex1^−/− mice in combination with both Nras^+/+ and Nras^Q61K/° transgenic modification. (b) Photomicrographs (H&E) of belly skin from the four genotypes represented in (a). Normal melanocyte situation in a C57BL6 mouse is in the hair follicles (black arrows). Melanocytes and deposition of melanin in the dermis (red arrow) and adipose tissue (red arrowheads) is seen in Nras^Q61K/° mice. Scale bars = 100µm.

Figure 2

P-Rex1^−/− mice have a defect in melanoblast migration. (a) RT-PCR of P-Rex1 mRNA in E14.5 embryo skin following FACS selection of cells enriched for melanoblasts (YFP+) compared to the rest of the embryo skin (YFP-). Tyrosinase and Dct controls confirm melanoblast enrichment. (b) E15.5 representative pictures comparing melanoblast migration to the belly in X-gal stained embryos. Scale bars = 500µm. (c) Comparison of melanoblast migration along forepaw in X-gal stained embryos at E15.5. A migratory deficit is evident in both P-Rex1^−/− (with either Nras^+/+ or Nras^Q61K/°) embryos. Scale bars = 500µm. (d) Schematic picture detailing quantification of melanoblast migration in the forepaw at E15.5. Levels 1 to 6 from top to bottom represent areas of 500µm × 500µm. Melanoblasts counted at each level and numbers compared between genotypes. (e,f) Comparison of melanoblast migration between P-Rex1^+/+ (blue bars) and P-Rex1^−/− (red bars) mice at levels 1 to 6 (as detailed in (d)). Upper panel (e) represents Nras^+/+ embryos (*p=0.01/0.02, Levels 2/3; Mann-Whitney test, n=5), lower panel (f) represents Nras^Q61K/° embryos (*p=0.02/0.02/0.01, Levels 1/2/3; Mann-Whitney test, n=5) (All box and whiskers plots: boxes represent 25^th-75^th percentiles of given value, lines represent median values) (g) Representative pictures at E15.5 of melanoblast morphology and protrusions (red arrows) on the flank of X-gal stained embryos. Scale bars = 100µm. (h) Quantification and comparison of percentage of cells with 1+ protrusions: P-Rex1^+/+ (N WT, blue bars) and P-Rex1^−/− mice (N HOM, red bars) (left hand panel; *p=0.05, Mann-Whitney; n=4); and Tyr::Nras^Q61K/°; P-Rex1^+/+ (Y WT, blue bars) and Tyr::Nras^Q61K/°; P-Rex1^−/− (Y HOM, red bars) mice (right hand panel; *p=0.03; Mann-Whitney, n=4). (i) Schematic picture detailing quantification of melanoblast cell numbers at E15.5. Areas represented: 1mm × 3mm. (j) Comparison of E15.5 melanoblast numbers between P-Rex1^+/+ (N WT, blue bars) and P-Rex1^−/− (N HOM, red bars) mice (left hand panel), and also between Tyr::Nras^Q61K/°; P-Rex1^+/+ (Y WT, blue bars) and Tyr::Nras^Q61K/°; P-Rex1^−/− (Y HOM, red bars) mice (right hand panel). (left panel: *p =0.01; right panel: *p =0.01; Mann-Whitney test, n=5)

Figure 3

P-Rex1^−/− mice have significantly reduced metastases in a Tyr::Nras^Q61K/°; INK4a^−/− murine melanoma model (a) Primary and metastatic melanomas arising in Tyr::Nras^Q61K/°; INK4a^−/− transgenic mice. Red arrowhead: pigmented primary skin melanoma; White arrowheads: multiple pigmented lung metastases; Red arrow: pigmented liver metastasis; White arrow: large pigmented brain lesion (b) MelanA and P-Rex1 immunohistochemical expression in primary melanomas of P-Rex1^+/+ mice. Varying levels of P-Rex1 expression can be seen in mouse melanomas (pink staining). n=10. (c) MelanA and P-Rex1 immunohistochemical expression in primary melanomas of P-Rex1^−/− mice. No P-Rex1 expression was observed in melanomas from these mice. n=9. (d) MelanA and P-Rex1 immunohistochemical expression in melanoma metastases from P-Rex1^+/+ control mice. Upper four panels show expression in brain lesions, while lower four panels show expression in lung metastases (pink staining). n=11. (e) Comparison of primary tumor burden between Tyr::Nras^Q61K/°; INK4a^−/−; P-Rex1^+/+ (blue bar) and Tyr::Nras^Q61K/°; INK4a^−/−; P-Rex1^−/− (red bar) mice (p=0.43, Mann-Whitney, n=29; small asterisks represent outlying data) (Box and whiskers plot: box represent 25^th-75^th percentile of given value, line represents median value). (f) Kaplan-Meier analysis of primary melanoma latency between Tyr::Nras^Q61K/°; INK4a^−/−; P-Rex1^+/+ (black line, n=27) and Tyr::Nras^Q61K/°; INK4a^−/−; P-Rex1^−/− (red line, n=18) cohorts (p=0.156, log-rank test). (g) Kaplan-Meier curves detailing significant improvement in survival of Tyr::Nras^Q61K/°; INK4a^−/−; P-Rex1^−/− cohort (red line) compared to Tyr::Nras^Q61K/°; INK4a^−/−; P-Rex1^+/+ cohort (black line) (*p=0.017, log-rank test, n=30 for each cohort).

Figure 4

P-Rex1 is upregulated in human melanoma where it drives invasion through its RacGEF activity (a) Western blots illustrating endogenous expression of P-Rex1 in a panel of human melanoma derived cell lines. Cell lines with driver mutations in NRAS and BRAF are represented, along with cell lines that are WT for both. NHM = normal human melanocytes. (b) P-Rex1 expression increases with more aggressive disease. Photomicrographs of human tissue specimens of normal skin, primary and metastatic melanoma, immunohistochemically labelled against P-Rex1 or the melanocyte marker melanA. LN = lymph node. Scale bars = 90µm. (c) Silencing P-Rex1 by siRNA as shown by western blot analysis. A mock transfection and scramble siRNA were used as controls. (d) Comparison of representative matrigel invasion assays of CHL1 human melanoma cell lines treated with control or P-Rex1 specific siRNA oligonucleotides. (e) Quantification of cells invading beyond 45µm in matrigel invasion assays of CHL1 human melanoma cell lines treated with control (‘siControl’) vs P-Rex1 (‘siP-Rex1’) specific siRNA oligonucleotides. (*p=0.005, Mann-Whitney test; n=3; error bars = +/− standard error of mean (SEM). (f) Westen blot confirming relative expression of ‘P-Rex1’, ‘P-Rex1 GD’, and ‘Tiam1’ cells is well matched. (g) Representative matrigel invasion assays comparing melanocytes derived from early pup skin of Tyr::Nras^Q61K/°; INK4a^−/−; P-Rex1^−/− mice and genetically modified to over-express P-Rex1 (‘P-Rex1’), empty vector (‘pLHCX’), GEF-dead P-Rex1 (‘P-Rex1 GD’), or Tiam1 (‘Tiam1’) (h) Left panel: quantification of cells invading beyond 45µm in matrigel invasion assays of pLHCX vs P-Rex1 vs P-Rex1 GD cells (*p=<0.0001 for both comparisons, Mann-Whitney test; n=3). Right panel: quantification of cells invading beyond 45µm in matrigel invasion assays of pLHCX vs P-Rex1 vs Tiam1 cells (*p=<0.0001 for both comparisons, Mann-Whitney test; n=3; error bars for both panels = +/− standard error of mean (SEM). (i) Representative organotypic invasion assays comparing Tyr::Nras^Q61K/°; INK4a^−/−; P-Rex1^−/− parental melanocytes genetically manipulated to over-express wild-type P-Rex1 (‘P-Rex1’), empty vector (‘pLHCX’) and GEF-dead P-Rex1 (‘P-Rex1 GD’) (n=3; Scale bars = 90µm).