Research ArticleCancer

Abl and Arg mediate cysteine cathepsin secretion to facilitate melanoma invasion and metastasis

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Science Signaling  20 Feb 2018:
Vol. 11, Issue 518, eaao0422
DOI: 10.1126/scisignal.aao0422
  • Fig. 1 Abl and Arg mRNAs are increased in melanoma cell lines and primary melanomas.

    (A) Abl/Arg mRNAs were quantitated with quantitative polymerase chain reaction (qPCR). Data are means ± SEM (n = 2), normalized to RPS13. (B) Relative mRNAs were plotted against protein levels (26) and subjected to linear regression. (C) Abl and/or Arg expression in primary melanomas and benign nevi (Nevi) reported in the Talantov et al. (29, 30) data set (melanoma, n = 45; nevi, n = 18) and deposited in Oncomine as well as The Cancer Genome Atlas (TCGA) data (melanoma, n = 103; metastases, n = 367) were reanalyzed using two-sample t tests (for normally distributed data, determined with Shapiro-Wilk test) or Wilcoxon rank sum tests. a.u., arbitrary units.

  • Fig. 2 Abl and Arg induce cathepsin secretion.

    (A and B) Western blotting of conditioned medium (CM) from cells transfected with two independent small interfering RNAs (siRNAs) (A and B) or a short hairpin RNA (shRNA) targeting Abl and Arg (B, right) and serum-starved for 16 hours. To adjust for differences in cell number, medium was loaded on the basis of cellular protein concentration. Lysate β-actin blots show loading, and β-actin blotting of media demonstrates media purity (lack of cell lysis). (C) Western blotting of media and lysate from serum-starved WM164 cells transiently transfected with constitutively active forms of Abl and/or Arg (PP). (D and E) Western blotting of conditioned medium from cells treated with Abl/Arg inhibitors nilotinib (Nilo; 435s, 5 μM; WM3248, 2 μM) or GNF-2 (GNF; 10 μM) during serum starvation (16 hours). Blots for pCrkL show efficiency of Abl/Arg inhibition by nilotinib and GNF-2. Blots are representative of three experiments. Means ± SEM are graphically shown in figs. S2 (A, D, and F) and S3A. CathL, cathepsin L; CathB, cathepsin B; Scr, scrambled control; Vec, control vector.

  • Fig. 3 Abl/Arg increase cathepsin proforms.

    (A to C) Western blotting of whole-cell lysates from siRNA-transfected (A and B), shRNA-transfected (B, right), or nilotinib-treated [(C); 435s, 5 μM; all other lines, 2 μM for 16 hours], serum-starved cells. pCrkL blots demonstrate efficiency of Abl/Arg inhibition by nilotinib (C). Blots are representative of three experiments. Means ± SEM are graphically shown in fig. S3 (C to E).

  • Fig. 4 Abl/Arg increase cathepsin mRNAs and transcription.

    (A and B) qPCR analysis of CTSL1 (CathL) and CTSB1 (CathB) mRNA abundance in 435s and WM3248 cells transfected with the indicated siRNA (A) or treated with nilotinib [(B); 435s, 5 μM; WM3248, 2 μM for 16 hours]. Data are means ± SEM normalized to expression of RPS13 (n = 3 experiments). ***P < 0.001, **P ≤ 0.01, and *P < 0.05 by one-sample t tests with Holm’s adjustment for multiple comparisons. Nilo, nilotinib; DMSO, dimethyl sulfoxide. (C and D) Gaussia luciferase activity was measured in the media of siRNA-transfected (C) or nilotinib-treated (D) cells stably expressing CathL or CathB promoter-luciferase constructs. Data are means ± SEM normalized to total protein in the lysate (n = 3 experiments). ***P < 0.001, **P ≤ 0.01, and *P < 0.05 by one-sample t tests with Holm’s adjustment for multiple comparisons. (E) CathB and/or CathL expression from data sets deposited in Oncomine (29, 30) were reanalyzed using two-sample t tests (for normally distributed data) or Wilcoxon rank sum tests. Nevi, benign nevi. Riker data set: melanoma, n = 14; metastases, n = 40; Talantov data set: melanoma n = 45; nevi = 18. (F) Analysis of normalized RNA sequencing (RNA-seq) data from the TCGA data set for correlation of CathL expression with that of Abl and/or Arg in primary melanomas (n = 103) and metastases (n = 367). Spearman’s correlation coefficient (r, with 95% confidence limits in parentheses) and P values are indicated.

  • Fig. 5 Abl and Arg promote expression of NF-κB (p65/RelA), Ets1, and Sp1.

    (A and B) Immunoblotting of whole-cell lysates (A) and subcellular fractions (B) from 435s and WM3248 cells transfected with control (Scr), Abl (#1), or Arg (#1) siRNA. (C and D) Cells treated with nilotinib (435s, 5 μM; WM3248, 2 μM, for 16 hours) were immunoblotted or subjected to subcellular fractionation as described for (A) and (B). pCrkL blots demonstrate efficiency of Abl/Arg inhibition by nilotinib. Blots are representative of three experiments. Means ± SEM are graphically shown in figs. S7 (A and B) and S8 (A and C). (E) Analysis of normalized RNA-seq data from the TCGA data set for correlation of Ets1 and Sp1 expression with that of Abl and Arg in primary melanomas (n = 103).

  • Fig. 6 NF-κB (p65/RelA), Ets1, and Sp1 transcription factors drive cathepsin expression in melanoma cells.

    (A) Western blotting of 435s or WM3248 cells transfected with control (Scr) or targeted siRNA (p65, Ets1, or Sp1). (B) qPCR for CathL or CathB expression in cells described in (A). (C) Gaussia luciferase assay from cells described in (A) cotransfected with luciferase reporters for the CathL or CathB promoter. (D) Western blotting of subcellular fractions from cells described in (A). Graphs are means ± SEM, relative to the control condition (Scr); n = 3 experiments. ***P < 0.001, **P ≤ 0.01, *P < 0.05 by one-sample t tests with Holm’s adjustment for multiple comparisons. (A and D) Blots are representative of n = 3 biological replicates. Means ± SEM are graphically shown in fig. S11 (A and E).

  • Fig. 7 Abl/Arg regulate cathepsin proform abundance through activation of NF-κB (p65/RelA).

    (A) Immunoblotting of lysates from 435s and WM3248 cells transiently transfected with vector or constitutively active IKKβ (IKKβ-EE) and control (Scr) or Abl- or Arg-targeted siRNA (#1, for 72 hours) and serum-starved. (B) Gaussia luciferase secretion measured in conditioned media from 435s cells expressing cathepsin L (CathL) or CathB promoter-luciferase constructs and transfected with siRNAs described in (A). Graphs are mean ± SEM, n = 3 experiments. ***P < 0.001, **P ≤ 0.01, *P < 0.05 by two-sample t tests with Holm’s adjustment for multiple comparisons. (C) Immunoblotting of lysates from 435s cells transfected with scrambled or one of two p65 siRNAs, treated with vehicle or nilotinib (5 μM for 16 hours). (D) Immunoblotting of lysates from nilotinib-treated 435s cells. pCrkL blots demonstrate efficiency of Abl/Arg inhibition by nilotinib. Blots are representative of three experiments. Means ± SEM are graphically shown in fig. S13 (A, D, and F).

  • Fig. 8 Abl/Arg drive invasion and metastasis by regulating cathepsin secretion.

    (A) Matrigel invasion assay of serum-starved 435s cells stably expressing vector or cathepsins B and L (CathB/L) and transfected with siRNA [control (Scr) or the siRNAs (#1) targeting Abl or Arg]. Cells were seeded on Matrigel in medium containing vehicle or cysteine cathepsin inhibitor E64C (50 μM) for 24 hours, above a compartment containing medium with 1% fetal bovine serum. An aliquot of cells was lysed and blotted, shown in fig. S14A. (B) Representative images from (A). Scale bars, 100 μm. (C) Matrigel invasion assay of WM3248 cells stably expressing vector or CathL that had been serum-starved and treated with nilotinib (2 μM for 16 hours) before seeding onto Matrigel-coated chambers over wells containing medium with IGF-1 (insulin-like growth factor 1) (10 nM). Invasion was assessed after 36 hours. An aliquot of cells was lysed and blotted, as shown in fig. S14B. Representative images are shown in fig. S14C. (A and C) Graphs are means ± SEM (n = 3 experiments). ***P < 0.001 and *P < 0.05 by two sample t tests with Holm’s adjustment for multiple comparisons. (D) Quantitation of green fluorescent protein–positive lung nodules from mice injected intravenously with vector- or CathL-expressing WM3248 cells and treated with vehicle or nilotinib for 33 days. For comparison, vector groups are shown on one scale (left), whereas all groups are shown on a different scale (right). Data are from n ≥ 11 mice; means are noted with a horizontal line. ***P < 0.001 by Wilcoxon rank sum test with Holm’s adjustment for multiple comparisons.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/518/eaao0422/DC1

    Fig. S1. Abl and Arg activities correlate with expression in melanoma cell lines.

    Fig. S2. Effects of silencing or inhibiting Abl/Arg on intracellular and extracellular cathepsins.

    Fig. S3. Abl/Arg drive cathepsin abundance.

    Fig. S4. Effects of silencing or inhibiting Abl/Arg on pro, intermediate, and mature (double-chain) cathepsin forms.

    Fig. S5. Abl and Arg promote cathepsin mRNA expression.

    Fig. S6. Abl/Arg and cathepsin mRNAs are correlated in primary melanomas.

    Fig. S7. Abl/Arg alter Ets1 and Sp1 protein, mRNA, and promoter activities.

    Fig. S8. Abl/Arg alter the nuclear localization of p65, Ets1, and Sp1.

    Fig. S9. Abl/Arg affect the DNA binding capacity of Ets1 and Sp1 on cathepsin promoters.

    Fig. S10. Abl/Arg and Sp1 or Ets1 expression is correlated in primary melanomas.

    Fig. S11. Ets1, Sp1, and p65 contribute to cathepsin abundance.

    Fig. S12. Model for Abl/Arg regulation of cathepsin expression and secretion.

    Fig. S13. Abl/Arg regulate cathepsin expression by activating NF-κB (p65).

    Fig. S14. Abl/Arg promote invasion by inducing cathepsin L secretion.

    Fig. S15. Abl/Arg promote lung colonization in a cathepsin L–dependent manner.

    Table S1. qPCR primer sequences.

    Table S2. ChIP primer sequences.

  • Supplementary Materials for:

    Abl and Arg mediate cysteine cathepsin secretion to facilitate melanoma invasion and metastasis

    Rakshamani Tripathi, Leann S. Fiore, Dana L. Richards, Yuchen Yang, Jinpeng Liu, Chi Wang, Rina Plattner*

    *Corresponding author. Email: rplat2{at}uky.edu

    This PDF file includes:

    • Fig. S1. Abl and Arg activities correlate with expression in melanoma cell lines.
    • Fig. S2. Effects of silencing or inhibiting Abl/Arg on intracellular and extracellular cathepsins.
    • Fig. S3. Abl/Arg drive cathepsin abundance.
    • Fig. S4. Effects of silencing or inhibiting Abl/Arg on pro, intermediate, and mature (double-chain) cathepsin forms.
    • Fig. S5. Abl and Arg promote cathepsin mRNA expression.
    • Fig. S6. Abl/Arg and cathepsin mRNAs are correlated in primary melanomas.
    • Fig. S7. Abl/Arg alter Ets1 and Sp1 protein, mRNA, and promoter activities.
    • Fig. S8. Abl/Arg alter the nuclear localization of p65, Ets1, and Sp1.
    • Fig. S9. Abl/Arg affect the DNA binding capacity of Ets1 and Sp1 on cathepsin promoters.
    • Fig. S10. Abl/Arg and Sp1 or Ets1 expression is correlated in primary melanomas.
    • Fig. S11. Ets1, Sp1, and p65 contribute to cathepsin abundance.
    • Fig. S12. Model for Abl/Arg regulation of cathepsin expression and secretion.
    • Fig. S13. Abl/Arg regulate cathepsin expression by activating NF-κB (p65).
    • Fig. S14. Abl/Arg promote invasion by inducing cathepsin L secretion.
    • Fig. S15. Abl/Arg promote lung colonization in a cathepsin L–dependent manner.
    • Table S1. qPCR primer sequences.
    • Table S2. ChIP primer sequences.

    [Download PDF]


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