Inactivation of the Hippo tumour suppressor pathway by integrin-linked kinase

Abstract

One of the hallmarks of cancers is the silencing of tumour suppressor genes and pathways. The Hippo tumour suppressor pathway is inactivated in many types of cancers, leading to tumour progression and metastasis. However, the mechanisms of pathway inactivation in tumours remain unclear. Here we demonstrate that integrin-linked kinase (ILK) plays a critical role in the suppression of the Hippo pathway via phospho-inhibition of MYPT1-PP1, leading to inactivation of Merlin. Inhibition of ILK in breast, prostate and colon tumour cells results in the activation of the Hippo pathway components MST1 and LATS1 with concomitant inactivation of YAP/TAZ (Yes-associated protein/transcriptional co-activator with PDZ-binding motif) transcriptional co-activators and TEAD-mediated transcription. Genetic deletion of ILK suppresses ErbB2-driven YAP/TAZ activation in mammary tumours, and its pharmacological inhibition suppresses YAP activation and tumour growth in vivo. Our data demonstrate a role for ILK as a multiple receptor proximal regulator of Hippo tumour suppressor pathway and as a cancer therapeutic target.

Figure 1. Silencing ILK leads to functional inactivation of YAP/TAZ in cancer cells.

Indicated cells were treated with non-silencing control (siCT) or two different siRNAs against ILK (siILK-A or siILK-H): (a) Cell lysates were subjected to western blotting with the indicated antibodies. Bands were semiquantified by image intensity area under the curve. (b) Cells were subjected to immunofluorescence microscopy with the indicated antibodies. Scale bar, 20 μm.

Figure 2. Pharmacological inhibition of ILK activates the Hippo pathway and suppresses YAP/TAZ activity in cancer cells.

Indicated cells were treated with DMSO (−) or the ILK inhibitor, QLT0267: (a) Cell lysates were subjected to western blotting with the indicated antibodies. Bands were semiquantified by image intensity area under the curve. (b) Cells were subjected to immunofluorescence microscopy with the indicated antibodies. Scale bar, 20 μm. (c) PC3 cells were incubated with DMSO (−) or QLT0267 followed by immunoprecipitation of endogenous TAZ (Ab) or an IgG control (IgG) and subjected to western blot analysis with the indicated antibodies. (d) TEAD transcriptional activity was assessed in the indicated cell lines by TEAD optimal and TEAD inactive mutant (CT)-dependent promoter-driven firefly luciferase reporter construct. Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=3 per group. (e) PC3 cells treated with the indicated concentrations of QLT0267 were counted daily to assess cell accumulation. Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=3 per group.

Figure 3. ILK is required for growth factor-mediated inactivation of the Hippo pathway.

Cells were treated with control (siCT) or ILK (siILK-A) or YAP (siYAP) or TAZ (siTAZ) siRNA, or pretreated with DMSO (−) or QLT0267 for 30 min: (a) Cells were serum-starved and incubated for 1 h with 100 ng ml⁻¹ of the indicated growth factors. Cell lysates were subjected to western blotting with the indicated antibodies. Bands were semiquantified by image intensity area under the curve. Numbers represent the ratio of each normalized phospho-Yap band intensity by total-Yap protein band intensity. (b,c) Cells were serum-starved, incubated with 5 ng ml⁻¹ TGF-β1 and subjected to immunofluorescence microscopy with the indicated antibodies. Scale bar, 20 μm. ILK depletion by western blot analysis is shown for BPH-1 cells. Arrows indicate nuclear localization. (d) Cell proliferation was assessed by MTT assay. Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=3 per group.

Figure 4. ILK inhibits the Hippo pathway through inactivation of Merlin by direct phosphorylation of MYPT1 phosphatase.

(a) Indicated cells were treated with DMSO (−) or ILK inhibitor, QLT0267, for the indicated periods of time. Cell lysates were analysed for phospho-Merlin expression by western blotting. Bands were semiquantified by image intensity area under the curve. (b) PC3 cells were treated as described above and analysed for phospho-MYPT1 expression by western blotting. Bands were semiquantified by image intensity area under the curve. (c) MDA-MB-435 LCC6 cells were incubated with DMSO (−) or QLT0267 and subjected to western blot analysis with the indicated antibodies.(d) Indicated cells were treated with DMSO (−), ILK inhibitor, QLT0267 or ROCK inhibitor, H1152, for the indicated periods of time. Hippo pathway components were analysed by western blotting using the indicated antibodies. (e) TEAD transcriptional activity was assessed by TEAD optimal and TEAD inactive mutant (CT)-dependent promoter-driven firefly luciferase reporter construct. Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=3 per group. (f) Indicated cells were treated with non-silencing control (siCT) or two different siRNAs against ILK (siILK-A or siILK-H) and cell lysates were subjected to western blotting with the indicated antibodies. (g) The indicated concentrations of GST-ILK were separated by SDS–polyacrylamide gel electrophoresis and subjected to Coomassie blue staining. (h) Western blot analysis of ILK phosphorylation of MYPT1 using various concentrations of ILK-GST (left panel) or reaction incubation times (right panel). Reactions carried out in the absence of GST-ILK are provided as negative controls. (i) Western blot analysis with the indicated antibodies assessing the kinase activity of ILK-GST towards MYPT1 with the indicated concentrations of QLT0267 inhibitor (left panel) or H1152 inhibitor (right panel).

Figure 5. ILK requires MYPT1 and Merlin to inactivate the Hippo pathway.

(a) PC3 cells were treated with non-silencing control (siCT) or siRNA against MYPT1 (siMYPT1) were incubated with DMSO or ILK inhibitor QLT0267 and cell lysates were subjected to western blotting with the indicated antibodies. Bands were semiquantified by image intensity area under the curve and represented as the ratio of each normalized phospho-antibody band intensity by total protein band intensity. Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=4 independent experiments. Not significant (n.s.). (b) Indicated cells were treated with DMSO or the ILK inhibitor QLT0267, fixed and subjected to immunofluorescence microscopy with the indicated antibodies. Scale bar, 20 μm. Right panels show Merlin expression in comparison with other cell lines by western blot analysis.

Figure 6. ILK inactivates the Hippo tumour suppressor pathway in vivo.

(a) Immunohistochemistry analysis of ErbB2-induced tumours with YAP/TAZ or an IgG control (IgG) antibody. Number of cells with nuclear and cytoplasmic (green, n=C), predominantly nuclear (red, N>C) and predominantly cytoplasmic (blue, C>N) YAP/TAZ localization were quantified in random 63 × views (right panel). Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=5 tumours per group. Lysates from tumours were subjected to western blotting to determine ILK expression (bottom right panel; 1=ILKwt/wt, 2=ILKf/f). (b,c) NIC WT cell lines established from the ErbB2 tumours were (b) treated with non-silencing control (siCT) or ILK siRNA (siILK-A), and subjected to immunofluorescence microscopy with the indicated antibodies. Scale bar, 20 μm. Or (c) treated with DMSO or ILK inhibitor QLT0267 and TEAD transcriptional activity was assessed by TEAD optimal and TEAD inactive mutant (CT)-dependent promoter-driven firefly luciferase reporter construct. Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=3 per group. (d) Immunohistochemistry analysis of Wnt1- and double-transgenic Wnt1/ILK-derived tumours with YAP/TAZ or an IgG control (IgG) antibody or ILK (1=Wnt, 2=Wnt/ILK). Number of cells with nuclear and cytoplasmic (green, n=C), predominantly nuclear (red, N>C) and predominantly cytoplasmic (blue, C>N) YAP/TAZ localization were quantified in random 63 × views (right panel). Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=4 tumours per group.

Figure 7. Pharmacological inhibition of ILK in vivo leads to decreased tumour growth and YAP/TAZ inactivation.

(a–e) Cells were treated with DMSO or ILK inhibitor QLT0267 at indicated concentrations: (a) TEAD transcriptional activity was assayed using TEAD optimal and TEAD inactive mutant (CT)-dependent promoter-driven firefly luciferase reporter construct. Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=3 per group. (b) Apoptosis was measured by TUNEL. Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=3 per group. (c) Cells were counted daily to assess cell accumulation. Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=3 per group. (d,e) Prior to treatment, cells were transfected with vector or constitutive active YAP (YAP S127A): (d) Apoptosis was measured by TUNEL. Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=3 per group. (e) Cells were counted daily to assess cell accumulation. Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=3 per group. (f) Luciferase-labelled MDA-MB-435 LCC6 cells were implanted orthotopically into Balb/c mice and treatment (Tx) was initiated 1 week later. Vehicle or 200 mg kg⁻¹ QLT0267 were administered daily by oral gavage and tumour growth was monitored by IVIS imaging. Error bars denote s.e.m. P-values were calculated using Student’s t-test; n=10–16 mice per group. (g) At the end of treatment, tumours were excised and subjected to immunohistochemistry with YAP/TAZ, or IgG control (IgG), or ILK antibodies. Representative images are shown. Arrows indicate YAP/TAZ nuclear localization. YAP/TAZ and IgG original magnifications × 63, ILK original magnification × 40.

Figure 8. Model for ILK regulation of the Hippo pathway.

PATHWAY OFF: ILK promotes tumour progression through phospho-dependent inactivation of MYPT1 phosphatase leading to inactivation of Merlin/MST/LATS key components of the Hippo pathway. PATHWAY ON: inhibition or genetic depletion of ILK results in the activation of the Hippo pathway resulting in the cytoplasmic retention and degradation of YAP/TAZ oncogenes and inhibition of tumour growth.