Loss of TGF-β tumour suppressive response is a hallmark of human cancers. As a central player in TGF-β signal transduction, SMAD4 (also known as DPC4) is frequently mutated or deleted in gastrointestinal and pancreatic cancer. However, such genetic alterations are rare in most cancer types and the underlying mechanism for TGF-β resistance is not understood. Here we describe a mechanism of TGF-β resistance in ALK-positive tumours, including lymphoma, lung cancer and neuroblastoma. We demonstrate that, in ALK-positive tumours, ALK directly phosphorylates SMAD4 at Tyr 95. Phosphorylated SMAD4 is unable to bind to DNA and fails to elicit TGF-β gene responses and tumour suppressing responses. Chemical or genetic interference of the oncogenic ALK restores TGF-β responses in ALK-positive tumour cells. These findings reveal that SMAD4 is tyrosine-phosphorylated by an oncogenic tyrosine kinase during tumorigenesis. This suggests a mechanism by which SMAD4 is inactivated in cancers and provides guidance for targeted therapies in ALK-positive cancers.
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RNA-Seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under the accession code GSE121188. Statistics source data for Figs. 3a–m,o, 4c–f, 6a–i and 7c,e–h and Supplementary Figs. 3a–c, 4b–d and 6b,c,e are provided in Supplementary Table 2. Unprocessed original scans of western blots are shown in Supplementary Fig. 8. All other data supporting the findings of this study are available from the corresponding author on reasonable request.
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We thank R. Chiarle for NPM–ALK and kinase-dead NPM–ALK(K210R), N. Fusenig for the HaCaT cell line, D. Luskutoff for p800(PAI-1)–Luc, Y. P. Mossé for the NB-1643 cell line, M. Vigny for the full-length ALK complementary DNA, B. Vogelstein for WWP1(p21)–Luc and SBE–Luc and X.-F. Wang for p15–Luc. We are indebted to L. Ding and Y. Ma at Betta Pharmaceutical for sharing the ALK inhibitor X396. We thank colleagues at Beijing Proteome Research Center and National Center for Protein Sciences (The PHOENIX Center, Beijing) for assistance with mass spectrometry analysis. We are grateful to K. Yu for sharing reagents, and J. Peng, Y. Zhu, N. Xu, H. Xia and members of the Feng laboratory for helpful discussion and technical assistance. We also thank K. Wrighton for editing the manuscript. We are grateful to L. Su for her contribution. This research was partly supported by grants from the NSFC (31730057, 91540205, 31090360, 31571447), the NIH (R21CA209007) and the DoD (DAMD W81XWH-15–1–0650/0651), and the Fundamental Research Funds for the Central Universities.
The authors declare no competing interests.
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Integrated supplementary information
(a) ALK interacts with SMAD4 under physiological conditions. Exponentially growing SUDHL-1 cells were treated with TGF-β and/or TAE684 for 3 h before harvest. Cell lysates were subject to IP-Western analysis using appropriate antibodies. The experiment was repeated three times independently with similar results. (b) SMAD4 directly binds to the kinase domain of ALK in vitro. GST-SMAD4 proteins were purified from E. coli BL21 (DE3) strain and mixed with in vitro synthesized FLAG-tagged NPM-ALK, NPM or ALK deletion mutants (as depicted in Figure 1e), respectively. GST-bound proteins and input were resolved by SDS-PAGE and detected by Western blotting. GST protein is used as a negative control. The experiment was repeated three times independently with similar results. (c) ALK directly binds to the MH1 domain of SMAD4. Purified GST-ALK-Δ2 proteins were mixed with in vitro synthesized HA-tagged SMAD4 deletion mutants respectively. GST-bound proteins and input were resolved by SDS-PAGE and detected by Western blotting. The experiment was repeated three times independently with similar results. Unprocessed blots are shown in Supplementary Fig. 8.
(a) Oncogenic ALK variants phosphorylate SMAD4. HEK293T cells were transfected with expression plasmids encoding FLAG-SMAD4 and ALK(WT), ALK(F1174L), NPM-ALK or EML4-ALK. Anti-FLAG IP-Western blotting analysis was carried out as described in Figure 2a. PY95 antibody was used to detect Y95 phosphorylation of SMAD4. The experiment was repeated three times independently with similar results. (b) Phosphorylation of GST-SMAD4 by immunopurified NPM-ALK. Recombinant NPM-ALK proteins immunopurified from transfected HEK293T cells were incubated with recombinant GST-SMAD4 purified from E. coli. In an in vitro kinase assay, the level of SMAD4 tyrosine phosphorylation was examined by Western blotting with PY100 antibody. The experiment was repeated three times independently with similar results. (c) NPM-ALK phosphorylates SMAD4 in the MH1 domain. NPM-ALK and FLAG-SMAD4 mutants were transfected into HEK293T cells. IP-Western blotting was carried out as described in Figure 2a. Domains of SMAD4 are described in Figure 1g. The experiment was repeated three times independently with similar results. (d) NPM-ALK phosphorylates SMAD4 in the MH1 domain. NPM-ALK and HA-SMAD4 truncations were transfected into HEK293T cells. The experiment was repeated three times independently with similar results. (e) NPM-ALK fails to phosphorylate the MH1 domain with Y95F substitution. HEK293T cells were transfected with NPM-ALK and FLAG-SMAD4MH1 (WT or Y95F). The experiment was repeated three times independently with similar results. (f) Depletion of NPM-ALK abolishes tyrosine phosphorylation of SMAD4 in Karpas 299 cells. Western blotting was done using appropriate antibodies. The experiment was repeated three times independently with similar results. Unprocessed blots are shown in Supplementary Fig. 8.
(a) NPM-ALK inhibits the TGF-β-induced transcriptional responses using reporter 3TP-Luc. HaCaT cells were transfected with expression plasmids for NPM-ALK or its kinase-dead K210R mutant, lucifersae reporter plasmid and Renilla luciferase. Relative luciferase activity was measured 18 h after TGF-β treatment (2 ng/ml). Values are normalized for transfection efficiency against Renilla luciferase activities. n = 3 independent experiments; Mean ± s.e.m. (b) NPM-ALK inhibits TGF-β-induced CAGA-Luc reporter response in HeLa cells. Assays were carried out as in panel a. n = 3 independent experiments; Mean ± s.e.m. (c) NPM-ALK inhibits TGF-β-induced SBE-Luc response in B lymphocyte BL41 cells. Assays were carried out as in panel a. n = 3 independent experiments; Mean ± s.e.m. (d) ALK(F1174L) blocks TGF-β-induced Caspase 3 activation. NMuMG cells (4x105) stably expressing ALK(F1174L) are treated with or without 10 ng/ml TGF-β for 24 h and then subjected to Western blotting. β-actin is a loading control. The experiment was repeated three times independently with similar results. (e) siRNA-mediated knockdown efficacy on NPM-ALK expression was measured in Karpas 299 cells. GAPDH is a loading control. The experiment was repeated three times independently with similar results. Unprocessed blots are shown in Supplementary Fig. 8. Statistical source data for panels a-c are provided in Supplementary Table 2.
Supplementary Figure 4 ALK inhibitor TAE-684 sensitizes ALK-positive tumour cells to TGF-β-induced target gene expression.
(a) SMAD4 knockout in Karpas 299 cells was demonstrated by anti-SMAD4 Western blotting. Clone 5 and 20 are two individual clones that were also confirmed by DNA sequencing. The experiment was repeated three times independently with similar results. (b) TAE-684 restores TGF-β-dependent CDKN1A transcription in H2228 cells. n = 3 independent experiments; Mean ± s.e.m. (c) TAE-684 restores TGF-β-dependent SERPINE1 transcription in Karpas 299 cells. n = 3 independent experiments; Mean ± s.e.m. (d) TAE-684 restores TGF-β-dependent SERPINE1 transcription in NB-1643 cells. n = 3 independent experiments; Mean ± s.e.m. Unprocessed blots are shown in Supplementary Fig. 8. Statistical source data for panels b-d are provided in Supplementary Table 2.
Supplementary Figure 5 ALK blocks the DNA-binding activity of SMAD4, but not its nuclear accumulation or SMAD complex assembly.
(a) Depletion of NPM-ALK has no effect on TGF-β-induced SMAD2/3 phosphorylation. H2228 cells were transfected with siRNA against ALK, treated with TGF-β (2 ng/ml, 1 h). Cell lysates were harvested for Western blotting analysis. The experiment was repeated three times independently with similar results. (b, c) Mutation at Y95 of SMAD4 has no effect on its interaction with SMAD2 (b) or SMAD3 (c). HEK293T cells were transfected with HA-SMAD2/SMAD3, FLAG-SMAD4 or a Y95 mutant (Y95E or Y95F), together with NPM-ALK (WT or K210R) and treated with TGF-β (2 ng/ml, 1 h). IP-Western blotting analysis was carried out. WT, wild-type; KR, K210R substitution; YE, Y95E substitution; YF, Y95F substitution. The experiments were repeated three times independently with similar results. (d, e) NPM-ALK represses the DNA binding of endogenous SMAD4. HaCaT-NPM-ALK(WT or K210R mutant) cells were treated with Dox and/or TGF-β (2 ng/ml, 1 h). DNA pull-down was done as described in Figure 5g. (d) Protein levels of SMAD4 and Dox-induced NPM-ALK (WT or K210R mutant) in whole cell lysates. (e) Protein levels of SMAD4 in DNA pull-down fractions. Biotin-SBEm, a biotin-labeled mutated SBE. The experiment was repeated three times independently with similar results. (f) SMAD4(Y95E) exerts a stronger interaction with p300 than wild-type SMAD4. The experiment was repeated three times independently with similar results. Unprocessed blots are shown in Supplementary Fig. 8.
(a-c) SMAD4(Y95E) fails to suppress tumour growth, while SMAD4(Y95F) exhibits the most potent tumour suppressor activity. SUDHL cells stably expressing GFP, SMAD4, SMAD4(Y95E) or SMAD4(Y95F) were injected subcutaneously into ancillary lymph nodes of nude mice to determine their tumorigenic activity. SUDHL-1 cells expressing SMAD4(Y95F) generate significantly smaller tumours than other groups as evaluated by tumour size (a), and weight (b). Panel c shows the time (days) of tumour appearance post injection. (b,c) n = 5 mice; Mean ± s.e.m.***, P < 0.001, **, P < 0.01, N.S., P > 0.05, by two-sided Student’s t test. The experiments were repeated three times independently with similar results. The data shown was obtained from one single experiment. (d, e) ALK inhibitor suppresses EAp9 lung tumour growth. EAp9 cells derived from EML4-ALK-induced mouse lung tumours were used to induce tumours in nude mice as described in panel a and treated with ALK inhibitor X396 at a dosage of 50 mg/kg every 2 days. Tumours were evaluated by tumour size (d) and weight (e). (e) n = 10 mice; Mean ± s.e.m. ***, P < 0.001, by two-sided Student’s t test. The experiment was repeated three times independently with similar results. The data shown was obtained from one single experiment. (f) ALK inhibitor blocks SMAD4 phosphorylation and enhances SMAD4 responses in EAp9-induced tumours. Tumour tissues from Panel d were subjected to Western blotting analysis. Phosphorylation levels of ALK and SMAD4 are indicated at top blots. The expression levels of PAI-1, p15Ink4B, p21Cip1 and MYC (SMAD4 target proteins), H3pS10 (proliferation index) and cleaved Caspase 3 (apoptosis index) are shown. The experiment was repeated three times independently with similar results. Unprocessed blots are shown in Supplementary Fig. 8. Statistical source data for panels b, c and e are provided in Supplementary Table 2.
(a) SMAD4 tyrosine phosphorylation at Y95 is correlated with the presence of NPM-ALK in human lymphoma. IHC staining of SMAD4, p-SMAD4(PY95) and ALK, as well as H&E staining are shown in one ALK-positive representative and one ALK-negative representative ALCL tumour sections. Pictures were taken under microscope in 10x, 20x, 40x, and 100x magnification, respectively. Scale Bars, 25 μm. Sections from 41 human patients were analyzed. One section for each patient was assessed and analyzed under microscope for five fields. (b) Y95 Phosphorylation is absent in Karpas 299(SMAD4-KO) tumours. To test the specificity of the PY95 antibody, IHC staining of SMAD4, PY95 and ALK was carried out in tumours derived from nude mice injected with Karpas 299 cells (2 samples) and Karpas 299(SMAD4-KO) (2 samples). Pictures were taken under microscope in 4x, 10x, 20x, and 40x magnification, respectively. Scale Bars, 50 μm. The experiment was repeated three times independently with similar results.
Unprocessed images of immunoblots shown in Figures and Supplementary Figures are provided.
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Zhang, Q., Xiao, M., Gu, S. et al. ALK phosphorylates SMAD4 on tyrosine to disable TGF-β tumour suppressor functions. Nat Cell Biol 21, 179–189 (2019). https://doi.org/10.1038/s41556-018-0264-3
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