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Human Cutaneous Melanomas Lacking MITF and Melanocyte Differentiation Antigens Express a Functional Axl Receptor Kinase  Marialuisa Sensi, Mara Catani,

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Presentation on theme: "Human Cutaneous Melanomas Lacking MITF and Melanocyte Differentiation Antigens Express a Functional Axl Receptor Kinase  Marialuisa Sensi, Mara Catani,"— Presentation transcript:

1 Human Cutaneous Melanomas Lacking MITF and Melanocyte Differentiation Antigens Express a Functional Axl Receptor Kinase  Marialuisa Sensi, Mara Catani, Giancarlo Castellano, Gabriella Nicolini, Federica Alciato, Gabrina Tragni, Giuseppina De Santis, Ilaria Bersani, Giancarlo Avanzi, Antonella Tomassetti, Silvana Canevari, Andrea Anichini  Journal of Investigative Dermatology  Volume 131, Issue 12, Pages (December 2011) DOI: /jid Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Expression of TAM RTKs in human melanoma lines and frequency distribution of Axl among tumors with different mutational status. (a) Axl mRNA levels were measured by real-time PCR in melanoma and human melanocyte lines (NHEM). Data are expressed as 2−ΔCt, where ΔCt indicates CT (Axl)-CT (β-actin). (b) Left-most column: frequency distribution of genetic alterations in the 58 human melanoma cell lines; right columns: frequency distribution of Axl in these melanomas, in the NRASQ61R or BRAFV600E subsets, and in melanomas negative for these mutations. Axl, Tyro3, and Mer expression measured by (c) real-time PCR with data presented as 2−ΔCt, where ΔCt indicates CT (target gene)-CT (β-actin); (d) immunoblot analysis where β-actin expression was used as a loading control. Molecular sizes of Axl, Mer, Tyro3, and β-actin were, respectively, 140, 170, 120, and 42kDa. Axlneg, Axl negative; Axlpos, Axl positive; NHEM, normal human epidermal neonatal melanocyte; RTK, receptor tyrosine kinase; TAM, Tyro3, Axl, Mer; wt, wild type. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Axl activation and autocrine production of Gas6. (a) Immunoblot of Akt and pAkt (both 57kDa) on lysates from Me#28 stimulated for specified times and doses with rGas6 with or without 1hour pretreatment with Axl/Fc (2.5μgml−1). (b) Phosphorylated Axl expression in Me#28, either untreated (-rGas6) or stimulated with rGas6 (500ngml−1 for 30minutes). Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody 4G10 and immunoblotting was performed with anti-Axl. (c) ELISA quantification of Gas6 levels in CM taken from confluent cultures of melanomas (empty bars) or from WI-38 (filled bar). Means±SD from two to four detections are shown. (d) ELISA quantification of Axl phosphorylation in lysates of Me#28 treated for 30minutes with CM from WI-38, Me#25 or Me#24, or with either CM preincubated for 1hour with Axl/Fc. CTRL−, no lysate; CTRL+, recombinant human phosphorylated Axl (2ngml−1). CM, conditioned medium; CTRL, control; IP, immunoprecipitation; pAkt, Akt phosphorylation; pTYR, phosphorylated tyrosines; WB, western blotting. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Cell-bound Gas6 expression in Axlpos melanomas. (a) Surface staining of Axl and Gas6 as detected by flow cytometry in six different melanoma cell lines. Gray histograms, staining with isotype control antibodies; dotted histograms, Axl expression; and bold histograms, Gas6 expression. (b) Axl knockdown abrogates cell-associated Gas6. Gray histograms, staining with isotype control antibodies; bold histograms, expression of Axl (left) or Gas6 (right) in Me#25 and 27 cells transfected with siAxl#3; and dotted histograms, expression of Axl (left) or Gas6 (right) in Me#25 and 27 cells transfected with siNeg#3. (c) Increased Gas6 secretion in CM from Axl-knockdown compared with parental and siNeg#3 Me#25 and 27 transfected cells as detected by ELISA. The numbers in histograms refer to MFI for Axl or Gas6 after subtraction of MFI of isotype control. Axlpos, Axl positive; CM, conditioned medium; MFI, median fluorescence intensity. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Graphical representation of the top score networks identified by IPA. Molecular relationships between genes correlating negatively (a, in green) or positively (b, in red) with Axl. Molecules and functions in networks: (A) local environment; (B) ECM interactions; (C) ECM modeling; (D) main ECM components; and (E) secreted factors. ECM, cell–extracellular matrix; IPA, Ingenuity Pathway Analysis. Gene names corresponding to gene symbols are listed in Supplementary Tables S5 and S6 online. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Expression of Axl is inversely correlated to that of MITF and MDAs. (a) MART-1, gp100, and MAGE-1 expression levels were examined by flow cytometry across a set of 10 Axlpos and 9 Axlneg melanoma samples. Isotype antibody was used as negative control. (b) Immunoblot analysis of Axl (140kDa), MITF (54kDa), and MART-1 (17kDa). β-Actin (42kDa) was used as a loading control. (c) Summary of immunohistochemical staining for Axl and MITF in melanoma specimens. (d) Staining for MITF and Axl in representative primary melanomas (#1–4). Me#4 is highly vascularized, and anti-Axl antibody stains endothelial cells as shown at higher magnification in the inset (arrow) and in Supplementary Figure S2 online. Bar=50μm. Axlneg, Axl negative; Axlpos, Axl positive; MFI, median fluorescence intensity; MITF, microphthalmia transcription factor. **P<0.01, ***P<0.001. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Relevance of Axl expression in motility and invasivity of human melanoma cells. (a) Invasion of Axlpos and Axlneg melanomas toward medium containing 10% FBS. The number of invading cells is reported as a percentage of seeded cells. White circle=Me#28. (b) Immunoblot of Axl, Akt, and pAkt in Axl-knockdown (siAxl#2, #3) or control (mock and siNeg#2, #3) Me#28 cells stimulated for 30minutes with 500ngml−1 rGas6. (c) Invasion, migration, and (d) wound healing of Axl-knockdown Me#28 cells in comparison with mock-transfected cells. Bar=200μm. (e) Wound closure of three different Axl-knockdown or control melanomas. (f) Migration of Axl-knockdown or control Me#28 cells through a HUVEC endothelial layer. Results are expressed as RFU values of migrated cells. (g) Wound healing, (h) migration, and (i) invasion of Me#28 in the presence of R428 (2μM) or vehicle (0.25% DMSO). Axlneg, Axl negative; Axlpos, Axl positive; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cell; pAkt, Akt phosphorylation; RFU, relative fluorescence unit. *P<0.05, **P<0.01. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

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