cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

Abstract

Inhibitor of apoptosis (IAP) proteins constitute a family of conserved molecules that regulate both apoptosis and receptor signaling. They are often deregulated in cancer cells and represent potential targets for therapy. In our work, we investigated the effect of IAP inhibition in vivo to identify novel downstream genes expressed in an IAP-dependent manner that could contribute to cancer aggressiveness. To this end, immunocompromised mice engrafted subcutaneously with the triple-negative breast cancer MDA-MB231 cell line were treated with SM83, a Smac mimetic that acts as a pan-IAP inhibitor, and tumor nodules were profiled for gene expression. SM83 reduced the expression of Snai2, an epithelial-to-mesenchymal transition factor often associated with increased stem-like properties and metastatic potential especially in breast cancer cells. By testing several breast cancer cell lines, we demonstrated that Snai2 downregulation prevents cell motility and that its expression is promoted by cIAP1. In fact, the chemical or genetic inhibition of cIAP1 blocked epidermal growth factor receptor (EGFR)-dependent activation of the mitogen-activated protein kinase (MAPK) pathway and caused the reduction of Snai2 transcription levels. In a number of breast cancer cell lines, cIAP1 depletion also resulted in a reduction of EGFR protein levels which derived from the decrease of its gene transcription, though, paradoxically, the silencing of cIAP1 promoted EGFR protein stability rather than its degradation. Finally, we provided evidence that IAP inhibition displays an anti-tumor and anti-metastasis effect in vivo. In conclusion, our work indicates that IAP-targeted therapy could contribute to EGFR inhibition and to the reduction of its downstream mediators. This approach could be particularly effective in tumors characterized by high levels of EGFR and Snai2, such as triple-negative breast cancer.

Fig. 1

Treatment with SM83 reduces the expression of Snai2 both in vivo and in vitro. a NOD/SCID mice engrafted subcutaneously with 5 × 10⁶ MDA-MB231 were treated with intraperitoneal injection of SM83 (5 mg/kg, 5 times/week) or left untreated (4 mice/group) until the end of the experiment. Schedule of the experiment (upper panel) and tumor volumes (bottom panel) measured twice a week (significant differences in days 24, 27, and 30. *P = 0.0476, 0.0391, and 0.0344, respectively. Unpaired two-tailed t-test). b Six hours after the last injection, mice were killed, nodules collected, and analyzed by western blot to detect the levels of SM83 targets cIAP1, cIAP2, and XIAP. Actin and vinculin are shown as loading controls. c MDA-MB231 cells transfected in vitro with two siRNAs targeting cIAP1 were treated or not with 100 nM SM83 for 1 h. Western blots were performed to evaluate the levels of cIAP1, cIAP2, and XIAP 72 h after transfection. Values show the fold levels of XIAP. d Differentially expressed genes: heat map showing the 50 genes significantly upregulated and the 15 downregulated by SM83 in MDA-MB231 nodules collected as described in Fig. 1a. e Wound-healing experiments performed with MDA-MB231 cells transfected with control (NT1) or Snai2-specific siRNAs (n = 4, **P = 0.0033. Unpaired two-tailed t-test). The graph shows the percentage of gap closure after 24 h of migration. The complete experiment with the other siRNAs tested is shown in Fig. S2a. f The levels of Snai2, downregulated in the GEP shown in Fig. 1d, and LRIG1, upregulated, were evaluated by western blot performed with lysates of MDA-MB231 nodules. Values show the fold levels of Snai2 and LRIG1 normalized to Actin levels. g Levels of Snai2 in MDA-MB231 cells treated with 100 nM SM83 in time-course experiments. Cleavage of p100 NF-kB2 into the p52 form was used to verify the expected activation of the non-canonical NF-kB pathway upon SM83 administration

Fig. 2

Snai2 expression is promoted by cIAP1, but not cIAP2 or XIAP. a Western blot showing the levels of Snai2 in MDA-MB231 cells transfected with siRNAs specific for cIAP1 or cIAP2, and, after 72 h, treated with 100 nM SM83 for further 6 h. b MDA-MB231 and BT549 cells were transfected as in Fig. 2a with siRNAs targeting XIAP and cIAP1, and western blot was performed to detect the levels of Snai2. c MDA-MB231, BT549, SUM159, and MDA-MB157 cells were transfected with two siRNAs specific for cIAP1 and the levels of Snai2 were detected. d The same cell lines employed in Fig. 2c were treated with 100 nM SM83 and viability tested with CellTiter-Glo assay 24 h later (two-tailed paired t-test, n = 3. MDA-MB231 **P = 0.0011; BT549 **P = 0.0082; SUM159 and MDA-MB157; ns not significant). e SM83 (100 nM) was used to treat MDA-MB231 and BT549 cells, pre-treated or not 1 h with 20 µM z-VAD, for 6 h and then cells were analyzed by western blot to detect the total levels of Snai2 and cIAP1, and the cleaved form of caspase-3. Values show the fold levels of Snai2 relative to untreated cells

Fig. 3

EGFR promotes Snai2 expression in a MAPK-dependent manner. a MDA-MB231, BT549, and MDA-MB157 cells were harvested after treatment for 2 h with 10 μM inhibitor of PI3K (LY294002), AKT (Triciribine), MEK (UO126), and p38 (SB203580). Western blot was performed to analyze the levels of Snai2. b MDA-MB231 cells were transfected with siRNAs specific for cIAP1, ERK1, and ERK2 to detect the levels of Snai2 72 h after transfection. cIAP1 and ERK1/2 are shown to control the silencing efficiency. c A panel of breast cancer cell lines was tested to compare the levels of Snai2. BaA basal “A”, BaB basal “B”, Lu Luminal [55]. d For each cancer type available in the TCGA study, Spearman’s correlation between EGFR and SNAI2 was calculated using RNA-Seq data (expressed as log2 counts per million mapped reads). Only primary tumors were considered in the analysis. Red arrow indicates the correlation bar in breast cancers. e BT549 cells were serum-starved overnight and then stimulated with 20 ng/ml EGF and TGFα in time-course experiments. Snai2 levels are shown together with total and activated levels of EGFR. MDA-MB231 (f) and BT549 (g) cells were serum-starved overnight, pre-treated or not with 100 μg/ml cetuximab for 1 h and then stimulated with 20 ng/ml EGF for the indicated time points. Western blot was performed to detect Snai2 levels, total ERK1/2 and EGFR, and their activated levels. Values show the fold levels of Snai2 relative to untreated cells. h Human mammary epithelial cell lines, parental and bearing mutated EGFR, were serum-starved and stimulated with 20 ng/ml EGF for the indicated times to evaluate Snai2 levels

Fig. 4

Depletion of cIAP1 hinders EGFR-dependent expression of Snai2. a MDA-MB231 and b BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, serum-starved overnight. Then, cells were stimulated for the indicated time points with 50 ng/ml and 20 ng/ml EGF, respectively. Levels of Snai2 and activated ERK1/2 were detected, together with cIAP1, to check the transfection efficiency. c BT549 and d MCF10A—wild-type or bearing mutated EGFR—cells were transfected as in Fig. 4a and stimulated with the indicated EGFR ligands (20 ng/ml) to evaluate the expression of Snai2 by western blot. e BT549 and f MCF10A cells were transfected and serum-starved as described before, stimulated 3 h with 20 ng/ml EGF and lysed to extract RNA. Real-time PCR was performed to evaluate Snai2 fold expression relative to GAPDH. BT549: *P = 0.0151, **P = 0.0036; n = 3; MCF10A: unstimulated siCtr vs. sicIAP1 *P = 0.0290, EGF 3 h siCtr vs. sicIAP1 *P = 0.0330; n = 5; two-tailed paired t-test

Fig. 5

Silencing of cIAP1 reduces EGFR levels. a BT549 and b MCF10A cells were transfected with control or cIAP1-specific siRNAs before being serum-starved overnight, stimulated with 20 ng/ml EGF and analyzed by western blot to detect the indicate proteins. c BT549 cells were transfected with control or cIAP1-specific siRNAs and, after 48 h, cells were serum-starved for 24 h and then stimulated 30 min with 20 ng/ml EGF. Fixed cells were incubated with anti-EGFR antibody and nuclei stained with DAPI. d LRIG1 expression levels were evaluated by real-time PCR in BT549 cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. Unstimulated siCtr vs. sicIAP1 *P = 0.0175, EGF 3 h siCtr vs. sicIAP1 *P = 0.0341; n = 3; two-tailed paired t-test. e Levels of Snai2 and LRIG1 in BT549 cells silenced for cIAP1 and stimulated or not with 20 ng/ml EGF. f EGFR levels in BT549 cells silenced for cIAP1 and LRIG1, stimulated or not with 20 ng/ml EGF. g LRIG1 expression levels were evaluated by real-time PCR in MCF10A cells serum-starved and stimulated with 20 ng/ml EGF after transfection with control or cIAP1-directed siRNAs. ns not significant; n = 4; two-tailed paired t-test

Fig. 6

cIAP1 reduces EGFR stability, but promotes its gene transcription. a EGFR and cIAP1 interaction was tested in BT549 cells using PLA assay. b BT549 cells stably expressing Myc/Flag-tagged EGFR were serum-starved and stimulated with 20 ng/ml EGF for the indicated times. Cells were lysed and EGFR was immunoprecipitated with anti-Flag antibody. Western blot was performed to evaluate the interaction of ectopic EGFR with cIAP1 and c-Cbl. c BT549 and MCF10A cells stably expressing Myc/Flag-tagged EGFR were serum-starved, pre-treated with 100 (BT549) or 50 (MCF10A) µg/ml cycloheximide for 30 min and stimulated with 20 ng/ml EGF for the indicated times. Levels of ectopic EGFR were detected with anti-Myc antibody. d Ectopic EGFR was immunoprecipitated as described in Fig. 6b from BT549 cells transfected with control and cIAP1-specific siRNAs. Western blot shows total levels of c-Cbl and the amount of c-Cbl interacting with EGFR. e BT549 and f MCF10A cell stably expressing ectopic Myc/Flag-tagged EGFR were further transduced with lentiviral particles to overexpress c-Cbl or GFP as a control. Cells were silenced for cIAP1, serum-starved, and stimulated with 20 ng/ml EGF as described above, and analyzed by western blot to evaluate the levels of ectopic EGFR by using a Myc-tagged-specific antibody. g BT549 (left panel) and MCF10A (right panel) were silenced for cIAP1 and analyzed by real-time PCR to quantify the levels of EGFR expression fold relative to GAPDH. BT549: unstimulated siCtr vs. sicIAP1 *P = 0.0134, EGF 3 h siCtr vs. sicIAP1 *P = 0.0270; n = 3. MCF10A: unstimulated siCtr vs. sicIAP1 ***P = 0.0004, EGF 3 h siCtr vs. sicIAP1 **P = 0.0183; n = 4; two-tailed paired t-test

Fig. 7

IAP inhibition hinders EGFR signaling independently from the receptor downregulation. a SUM159 and b MCF10A cells were serum-starved, pre-treated or not with SM83, and stimulated with 20 ng/ml EGF for the indicated times. Western blots were performed to evaluate the total and activated levels of EGFR and ERK1/2, and total levels of Snai2. cIAP1 is shown to control the efficiency of the treatment. c MCF10A cell viability was tested by CellTiter-Glo 24 h after treatment with 100 nM SM83. d BT549 cells ectopically expressing Myc/Flag-tagged EGFR, silenced with control and cIAP1-specific siRNAs, were serum-starved overnight and then stimulated with 20 ng/ml EGF. Western blot was performed to detect the total levels of ectopic EGFR (Myc), cIAP1, and Snai2. e BT549 cells ectopically expressing Myc/Flag-tagged EGFR were serum-starved, pre-treated or not 1 h with SM83, and stimulated with 20 ng/ml EGF for the indicated times. Western blot was performed to detect the total levels of ectopic EGFR (Myc), ERK1/2, cIAP1, and Snai2, and the activated form of ERK1/2. f Tumors described in Fig. 1b were analyzed by western blot to detect the activation of ERK1/2, and the total levels of EGFR and ERK1/2

Fig. 8

SM83 treatment results in anti-tumor and anti-metastasis effect. a Lungs of NOD/SCID mice bearing MDA-MB231 tumors were collected 2 weeks after the last injection with SM83 (upper panel, see Fig. S4a for primary tumor volumes), formalin-fixed and paraffin-embedded, and stained with an anti-human vimentin antibody to detect spontaneous metastasis (bottom panel). b Number (untreated n = 7, SM83-treated mice n = 8; sum of two independent experiments shown in Fig. S4a–b; *P = 0.0238. Unpaired two-tailed t-test) and c size (35 metastases/group; *P = 0.0107. Unpaired two-tailed t-test) of spontaneous MDA-MB231 lung metastases were evaluated. d Western blot showing the levels of cIAP1, EGFR, ERK1/2, pERK1/2, and Snai2 in primary tumors at the end of the experiment described in Fig. 8a