TNFα-YAP/p65-HK2 axis mediates breast cancer cell migration

Abstract

Clinical and experimental evidence indicates that macrophages could promote solid-tumor progression and metastasis. However, the mechanisms underlying this process remain unclear. Here we show that yes-associated protein 1 (YAP1), a transcriptional regulator that controls tissue growth and regeneration, has an important role in tumor necrosis factor α (TNF α)-induced breast cancer migration. Mechanistically, macrophage conditioned medium (CM) or TNFα triggers IκB kinases (IKKs)-mediated YAP phosphorylation and activation in breast cancer cells. We further found that TNFα or macrophage CM treatment increases the interaction between p65 and YAP. Chromatin immunoprecipitation (ChIP) assay shows that YAP/TEAD (TEA domain family member) and p65 proteins synergistically regulate the transcription of hexokinase 2 (HK2), a speed-limiting enzyme in glycolysis, and promotes TNFα-induced or macrophage CM-induced cell migration. Together, our findings indicate an important role of TNFα-IKK-YAP/p65-HK2 signaling axis in the process of inflammation-driven migration in breast cancer cells, which reveals a new molecular link between inflammation and breast cancer metastasis.

Figure 1

Macrophage CM promotes cell migration and YAP activation. (a) Control or shYAP stably transfected MCF7 cells were cultured with control medium or macrophage CM in the transwells for 24 h. The migratory ability was determined by transwell assay. The right panel showed the YAP knockdown efficiency. (b) The transwell membranes were washed by dimethylsulfoxide (DMSO) and the OD values were calculated at the absorbance of 570 nm. Data were collected from three independent experiments. (c, d) MCF7 cells were cultured in control medium or macrophage CM for 24 h, YAP expression was detected by western blot (c) and real-time PCR (d). (e) Control or shYAP stably transfected MCF7 cell lines were cultured with macrophage CM for 24 h and then the CYR61 mRNA level was assessed via real-time PCR. The error bars represent the means±s.d. (NS, no significance; **P<0.01; ***P<0.001, n=3).

Figure 2

TNFα increases the activation of YAP in breast cancer cells. (a) MCF7 cells were cultured with control medium or macrophage CM, and simultaneously treated with 0.5 μM Src-inhibitor saracatinib, 10 μM JAK-inhibitor tofacitinib, 10 μM mitogen-activated protein kinase-inhibitor SB203580 or 10 μM IKK-inhibitor IKK-16 for 24 h, the cells were lysed and YAP protein level was monitered by western blot. (b) Control or shYAP MCF7 cell lines were treated with 10 μM TNFα for 24 h and the cell migration was demonstrated by transwell assay, the results were shown as the invasive cell OD value. Data were collected from at least three independent experiments. (c) MCF7 cells were treated with 10 μM TNFα for 24 h, the cell lysates were subject to western blot to detect YAP expression. (d) MCF7 cells were treated with 10 μM TNFα for 12 h, YAP mRNA level was measured by real-time PCR. (e) MCF7 cells were treated with 10 μM TNFα for 12 h and the mRNA levels of CTGF and CYR61 were measured by real-time PCR. (f, g) Control and shYAP MCF7 cells were treated with 10 μM TNFα for 12 h. Lactate production (f) and glucose consumption (g) were measured. The error bars represent the means±s.d. (**P<0.01; ***P<0.001; n=3).

Figure 3

YAP mediates TNFα-induced expression of HK2 and cell migration in breast cancer cells. (a, b) Control and shYAP MCF7 cells were treated with 10 μM TNFα for 12 h or 24 h. HK2 mRNA and protein levels were detected via real-time PCR (a) and western blot (b). (c, d) Control and shYAP MCF7 cells were cultured with control medium or the indicated macrophage CM for 12 h. HK2 mRNA and protein levels were determined by real-time PCR (c) and western blot (d). (e) Control and shTEAD MCF7 cells were cultured in control medium or macrophage CM. The protein level of HK2 was determined by western blot. (f) MCF7 cells were transfected with siRNA for TNFR1 and then cultured in macrophage CM or treated with 10 μM TNFα. The cells were lysed for the detection of HK2 expression. (g, h) MCF7 cells stably expressing shRNA targeting HK2 were cultured in macrophage CM (g) or treated with 10 μM TNFα (h) in transwells for 24 h. Cell migration were analyzed by transwell assay. (i) Clinical specimens of breast cancer were immunostained with antibodies against CD68, YAP and HK2, the typical specimens of the same region were exhibited, the classification and the correlation between two proteins were performed. The error bars represent the means±s.d. (**P<0.01; ***P<0.001; n=3).

Figure 4

IKKβ and IKKε phosphorylate YAP. (a) 293T cells were co-transfected with GFP-YAP and Flag-IKKα, HA-IKKβ or IKKε. The cell lysates were analyzed by western blot with indicated antibodies. The asterisk showed the shifted band (b, c) 293T cells were co-transfected with GFP-YAP and HA-IKKβ or IKKε. Cell lysates were immunoprecipitated with indicated antibodies and analyzed by western blot. (d) 293T cells were transfected with GFP-YAP and HA-IKKβ or IKKε. The cell lysates were analyzed by phos-tag gel western blot. The asterisk showed the phosphorylated band. (e) The recombinant GST-YAP protein was incubated with immunoprecipitated HA-IKKβ or IKKε in phosphorylation buffer. Reactions were subjected to electrophoresis and immunoblotted with antibody against pan-phosphorylated serine/threonine (S/T). (f) MCF7 cells were treated with TNFα for indicated times, the cells were then lysed for phos-tag gel western blot. The asterisk showed the phosphorylated band. (g) 293T cells were transfected with GFP-YAP and HA-IKKβ/ε. Cell lysates were immunoprecipitated with GFP antibody followed by immunoblotting with TEAD4 antibody. (h) MCF7 cells were treated with 10 μM TPCA-1 or 10 μM IKK-16 for 12 h. Then endogenous YAP was immunoprecipitated and followed by immunoblotting with TEAD4 antibody.

Figure 5

YAP-TEAD and p65 synergistically regulate the expression of HK2. (a) MCF7 cell lines stably expressing shRNA (1# and 2#) targeting p65 were treated with control or 10 μM TNFα or macrophage CM for 24 h, p65 knockdown efficiency and HK2 protein level were confirmed by western blot. (b) 293T cells were co-transfected with GFP-YAP and Myc-p65. Cell lysates were immunoprecipitated with Myc antibody and then analyzed by western blot. (c) 293T cells were co-transfected with GFP-p65 and Flag-YAP. Cell lysates were immunoprecipitated with Flag antibody and then analyzed by western blot. (d) 293T cells were transfected with Myc-p65 and HA-TEAD4. Immunoprecipitation of Myc-p65 and co-immunoprecipitation of HA-TEAD4 were detected by western blot. (e) MCF7 cells were treated with 10 μM TNFα for 12 h, cell lysates were immunopreipitated with rabbit IgG or YAP antibody, the endogous co- immunopreipitated p65 was measured by immunoblotting. (f) 293T cells were co-transfected with GFP-YAP and Myc-p65. After transfection, the cells were treated with 10 μM TPCA-1 or 10 μM IKK-16 for followed 18 h, Myc-p65 was immunoprecipitated with Myc antibody and the co-immunoprecipitated GFP-YAP was determined by western blot. (g) MCF7 cells were treated with 10 μM TNFα for 12 h. The cell lysates were used for ChIP analysis with antibody against TEAD4, YAP or p65. The binding of TEAD4, YAP or p65 on HK2 promoter were detected by real-time PCR. (h, i) Control and shYAP MCF7 cells were treated with TNFα for 12 h. The mRNA levels of CCL2 (h) and IL-6 (i) were analyzed by real-time PCR. (j, k) MCF7 cells were treated with 10 μM TNFα for 12 h. Then, the cell lysates were used for ChIP analysis with antibody against TEAD4, YAP or p65. The binding of TEAD4, YAP or p65 on CCL2 or IL-6 promoter were detected by real-time PCR. (l) The model of YAP/TEAD/p65 interact with each other to synergistically regulate gene transcription and breast cancer cell migration under TNFα treatment. The error bars represent the means±s.d. (*P<0.05; **P<0.01; ***P<0.001, n=3).