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The E3 ligase APC/CCdh1 promotes ubiquitylation-mediated proteolysis of PAX3 to suppress melanocyte proliferation and melanoma growth

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Science Signaling  01 Sep 2015:
Vol. 8, Issue 392, pp. ra87
DOI: 10.1126/scisignal.aab1995
  • Fig. 1 Correlation between Cdh1 and PAX3 abundance in melanoma.

    (A and B) Immunohistochemistry (A) and quantification (B) of Cdh1 staining (brown) in human skin and melanoma tissue spanning the benign to metastatic pathological stages. (C) Immunohistochemical analysis of the correlation between Cdh1 and PAX3 staining in patient primary melanoma tissue.

  • Fig. 2 APCCdh1 negatively regulates PAX3 abundance in melanocytes.

    (A and B) Immunoblot (IB) analysis of whole-cell lysates (WCL) derived from mouse melan-a cells (A) or human primary melanocytes (HPM) (B) infected with species-specific Cdh1 shRNAs. (C and D) Immunoprecipitation (IP) for hemagglutinin (HA) and immunoblot for Flag in human primary melanocytes (C) or melan-a (D) cells transfected with HA-ubiquitin, the indicated Flag-PAX3, and wild-type (WT) or mutant Myc-Cdh1 constructs and treated with MG132 (25 μM, 6 hours). (E and F) Immunoblot of whole-cell lysates derived from melan-a (E) or human primary melanocytes (F) transfected with PAX3 and WT or mutant Cdh1. (G and H) As in (A) and (B) in the presence of MG132 (10 μM, 10 hours) where indicated. (I to N) Immunoblot analysis of melan-a or human primary melanocytes infected with shRNAs against Cdc27 (I and J), APC10 (K and L), or Cdc20 (M and N). (O and P) Immunoblot analysis in human primary melanocytes (O) or melan-a cells (P) infected with Cdh1 shRNA or a negative control (Scr) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing cycloheximide (CHX). Blots are representative of three experiments.

  • Fig. 3 Cdh1, but not Cdc20, specifically interacts with PAX3 to promote its degradation in a D-box–dependent manner.

    (A and B) Immunoblotting for endogenous Cdh1 and PAX3 in whole-cell lysates after immunoprecipitation in melan-a (A) or human primary melanocytes (B) pretreated with MG132 (10 μM, 10 hours). Immunoglobulin G (IgG) served as the immunoprecipitation control. (C) Immunoprecipitation and immunoblotting in whole-cell lysates (WCL) from 293T cells transfected with Flag-PAX3 and HA-Cdh1 or HA-Cdc20, pretreated with MG132 (10 μM, 10 hours). (D and E) Immunoblotting of whole-cell lysates from melan-a (D) or human primary melanocytes (E) transfected with PAX3 and the indicated HA-Cdc20 or HA-Cdh1 constructs. (F) GST pull-down analysis to assess GST-Cdh1 interaction with Flag-PAX3 in transfected 293T cells. (G and H) Immunoblotting for Flag (PAX3), HA (Cdh1), and cyclin B in melan-a cells transfected with Flag-tagged WT (G) or 3D-box mutant (H) PAX3 with increasing abundance of ectopic HA-tagged Cdh1. (I) Immunoprecipitation and immunoblotting in whole-cell lysates from 293T cells transfected with HA-Cdh1 and the indicated PAX3 construct, pretreated with MG132 (10 μM, 10 hours). (J) Immunoprecipitation and immunoblotting analysis for endogenous Cdh1 in whole-cell lysates from B16 cells transfected with the indicated Flag-PAX3 construct. (K) GST pull-down analysis to assess GST-Cdh1 interaction with WT or D-box mutant His-PAX3 in transfected 293T cells. Blots are representative of three experiments.

  • Fig. 4 APC/CCdh1 promotes the ubiquitylation of PAX3 in a D-box–dependent manner.

    (A) Immunoblot analysis of whole-cell lysates from 293T cells transfected with HA-Cdh1 and the indicated full-length (FL) or mutant Flag-PAX3 construct. (B) Immunoblot of whole-cell lysates from 293T cells transfected with Flag-PAX3 together with the indicated HA-Cdh1 constructs. EV, empty vector. WT, amino acids 1 to 496; N/F1, amino acids 1 to 395; N/F2, amino acids 1 to 305; N, amino acids 1 to 155. (C and D) Pull-down (IP) for Flag-PAX3 and immunoblotting for HA-ubiquitin in transfected mouse melan-a (C) or human primary melanocytes (D) infected with control or Cdh1 shRNA and pretreated with MG132 (25 μM, 6 hours). (E) As in (C) in control or Cdh1-depleted melan-a cells transfected with HA-ubiquitin and full-length or D-box mutant Flag-PAX3, pretreated with MG132. (F) In vitro ubiquitination assay using WT or 3D-box mutant His-PAX3. (G) Immunoprecipitation with Ni-NTA–conjugated agarose beads followed by immunoblotting as indicated in 293T cells transfected with His-ubiquitin, Flag-PAX3, and WT or mutant HA-Cdh1 (or an empty vector), and pretreated with MG132. Blots are representative of three experiments.

  • Fig. 5 Ectopic overexpression of Cdh1 inhibits melanocyte proliferation.

    (A and B) Relative cell number (A) and cell cycle profiles (B) in human primary melanocytes infected with HA-tagged Cdh1 or a control viral vector. *P < 0.01 by t test. (C and D) As in (A) and (B) in cells overexpressing both HA-Cdh1 and HA-PAX3. *P < 0.01 by t test. (E) Immunoblotting in whole-cell lysates from human primary melanocytes before and 1 to 6 hours after irradiation with UVB. Blot is representative of three independent experiments. Data are means ± SD of three experiments.

  • Fig. 6 APC/CCdh1 functions in melanin production.

    (A to C) Immunoblot (A), melanin production (B), and tyrosinase activity (C) in murine B16 melanoma cells infected with lentiviral shRNA against Cdh1 or a negative control (shScr). Tyr, tyrosinase. (D to F) As in (A) to (C) in human A375 melanoma cells. (G to I) Immunoblot (G), melanin production (H), and tyrosinase activity (I) in B16 cells transfected with HA-tagged empty vector, WT Cdh1, or mutated Cdh1 (ΔC-box). (J to L) As in (G) to (I) in A375 cells. Data are means ± SD from three independent experiments.

  • Fig. 7 Cdh1 deficiency promotes melanomagenesis.

    (A) Immunoblot analysis of whole-cell lysates from A375 cells infected with the indicated lentiviral constructs. (B and C) A375 cells were subjected to colony formation (B) and soft agar (C) assays of A375 cells generated in (A) cultured for 21 days. Data are means ± SD from three independent experiments. (D and E) Tumor mass of A375 (D) or UACC62 (E) melanoma xenografts that were infected with shRNA against CDH1 or a negative control before subcutaneous inoculation. Tumor mass was measured at day 34 after injection. Data are means ± SEM from 10 tumors. *P < 0.05 (Student’s t test). (F and G) Tumor mass of A375 (F) or B16 (G) melanoma xenografts that were infected with targeted or control shRNA as described in (D) and (E). Data are means ± SEM from 10 tumors. *P < 0.05, **P < 0.001 (Student’s t test).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/392/ra87/DC1

    Fig. S1. Correlation and colocalization between Cdh1 and PAX3 abundance in normal skin and melanoma tissue.

    Fig. S2. Depletion of endogenous Cdh1 does not affect PAX3 mRNA expression.

    Fig. S3. Detection of colocalization between Cdh1 and PAX3 in melanocytes and melanoma cell lines.

    Fig. S4. APC/CCdh1 E3 ligase does not target MITF in melanocytes.

    Fig. S5. Interaction with PAX3 is impaired by the expression of melanoma-derived Cdh1 mutants.

    Fig. S6. Depletion of endogenous Cdh1 in mouse and human melanocytes increases MITF and tyrosinase signaling.

    Fig. S7. Cdh1 silencing decreases doxorubicin sensitivity in melanoma cells.

    Table S1. Genetic mutations of APC/C components in TCGA melanomas and exome sequencing data.

  • Supplementary Materials for:

    The E3 ligase APC/CCdh1 promotes ubiquitylation-mediated proteolysis of PAX3 to suppress melanocyte proliferation and melanoma growth

    Juxiang Cao, Xiangpeng Dai, Lixin Wan, Hongshen Wang, Jinfang Zhang, Philip S. Goff, Elena V. Sviderskaya, Zhenyu Xuan, Zhixiang Xu, Xiaowei Xu, Philip Hinds, Keith T. Flaherty, Douglas V. Faller, Colin R. Goding, Yongjun Wang,* Wenyi Wei,* Rutao Cui*

    *Corresponding author. E-mail: yjwang88{at}hotmail.com (Y.W.); wwei{at}bidmc.harvard.edu (W.W.); rutaocui{at}bu.edu (R.C.)

    This PDF file includes:

    • Fig. S1. Correlation and colocalization between Cdh1 and PAX3 abundance in normal skin and melanoma tissue.
    • Fig. S2. Depletion of endogenous Cdh1 does not affect PAX3 mRNA expression.
    • Fig. S3. Detection of colocalization between Cdh1 and PAX3 in melanocytes and melanoma cell lines.
    • Fig. S4. APC/CCdh1 E3 ligase does not target MITF in melanocytes.
    • Fig. S5. Interaction with PAX3 is impaired by the expression of melanoma-derived Cdh1 mutants.
    • Fig. S6. Depletion of endogenous Cdh1 in mouse and human melanocytes increases MITF and tyrosinase signaling.
    • Fig. S7. Cdh1 silencing decreases doxorubicin sensitivity in melanoma cells.
    • Table S1. Genetic mutations of APC/C components in TCGA melanomas and exome sequencing data.

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    Citation: J. Cao, X. Dai, L. Wan, H. Wang, J. Zhang, P. S. Goff, E. V. Sviderskaya, Z. Xuan, Z. Xu, X. Xu, P. Hinds, K. T. Flaherty, D. V. Faller, C. R. Goding, Y. Wang, W. Wei, R. Cui, The E3 ligase APC/CCdh1 promotes ubiquitylation-mediated proteolysis of PAX3 to suppress melanocyte proliferation and melanoma growth. Sci. Signal. 8, ra87 (2015).

    © 2015 American Association for the Advancement of Science

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