EDIL3 promotes epithelial–mesenchymal transition and paclitaxel resistance through its interaction with integrin αVβ3 in cancer cells

Gasca et al. Cell Death Discovery (2020) 6:86 https://doi.org/10.1038/s41420-020-00322-x Cell Death Discovery ARTICLE Open Access EDIL3 promotes epithelial–mesenchymal transition and paclitaxel resistance through its interaction with integrin α V β 3 in cancer cells J. Gasca 1 ,M.L.Flores 1 , R. Jiménez-Guerrero 1 ,M.E.Sáez 2 , I. Barragán 3,4 ,M.Ruíz-Borrego 5 ,M.Tortolero 6 , F. Romero 6 ,C.Sáez 1,7 andM.A.Japón 1,7 Abstract Epithelial–mesenchymal transition (EMT) has recently been associated with tumor progression, metastasis, and chemotherapy resistance in several tumor types. We performed a differential gene expression analysis comparing paclitaxel-resistant vs. paclitaxel-sensitive breast cancer cells that showed the upregulation of EDIL3 (EGF Like Repeats and Discoidin I Like Domains Protein 3). This gene codifies an extracellular matrix protein that has been identified as a novel regulator of EMT, so we studied its role in tumor progression and paclitaxel response. Our results demonstrated that EDIL3 expression levels were increased in paclitaxel-resistant breast and prostate cancer cells, and in subsets of high-grade breast and prostate tumors. Moreover, we observed that EDIL3 modulated the expression of EMT markers and this was impaired by cilengitide, which blocks the EDIL3–integrin α V β 3 interaction. EDIL3 knockdown reverted EMT and sensitized cells to paclitaxel. In contrast, EDIL3 overexpression or the culture of cells in the presence of EDIL3enriched medium induced EMT and paclitaxel resistance. Adding cilengitide resensitized these cells to paclitaxel treatment. In summary, EDIL3 may contribute to EMT and paclitaxel resistance through autocrine or paracrine signaling in cancer cells. Blockade of EDIL3–integrin α V β 3 interaction by cilengitide restores sensitivity to paclitaxel and reverts EMT in paclitaxel-resistant cancer cells. Combinations of cilengitide and taxanes could be beneficial in the treatment of subsets of breast and prostate cancers. Background Epithelial–mesenchymal transition (EMT) is the process by which epithelial cells loose the adherent and tight junctions that keep them in contact with their neighbor cells to gain a mesenchymal phenotype. This transition favors an increased mobility, migration, or invasion. EMT is a complex process involving several transcription factors, cell-surface and cytoskeletal proteins, components of the extracellular matrix (ECM), and numerous signaling pathways, including Wnt/β-catenin, TGF-β, Hedgehog, and integrin pathways 1,2 . Among the transcriptional factors involved, members of the Snail family (SNAI1, SNAI2/Slug, and Twist) are considered to be key regulators of EMT. Concretely, SNAI1 directly represses Ecadherin expression that provides the physical structure for both cell–cell junctions and for the recruitment of signaling complexes in epithelial cells 2,3 . Thus, the loss of E-cadherin expression is one of the earliest events in the EMT process and, together with the gain of vimentin expression, is commonly used to demonstrate EMT in experimental situations 1,4 . Although EMT is a key process during embryogenesis, wound healing and tissue regeneration, it has recently been suggested to play an important role in tumor progression and metastasis 1,2,4 .In breast cancer, EMT is associated with unfavorable © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Correspondence: C. Sáez ([email protected]) or M. A. Japón ([email protected]) 1 Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, 41013 Seville, Spain 2 Centro Andaluz de Estudios Bioinformáticos (CAEBi), 41013 Seville, Spain Full list of author information is available at the end of the article These authors contributed equally: J. Gasca, M. L. Flores These authors jointly supervised this work: C. Sáez, M. A. Japón Edited by Alessandro Rufini Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; prognosis and metastasis, and it has been proposed that EMT can be responsible for the spreading of breast epithelial tumor cells to the liver, lungs, or bone marrow 5,6 . Moreover, resistance to paclitaxel, docetaxel, doxorubicin, and tamoxifen has been linked with the aberrant expression of the EMT-related genes Twist, Snail, and Slug, as well as with the induction of EMT in breast cancer cells 7–10 . Finally, EMT has been associated with the aggressive behavior and resistance to docetaxel therapy in advanced prostate cancer 11,12 . EGF Like Repeats and Discoidin I Like Domains Protein 3 (EDIL3), also known as Developmental Endothelial Locus 1, is an ECM protein containing three EGF-like domains; the second one has a RGD motif (Arg–Gly–Asp) that allows the interaction of EDIL3 with integrins. EDIL3 acts as a pro-angiogenic factor, mediator of the immune and anti-inflammatory response, and a regulator of endothelial cell adhesion and migration 13–15 . It has been reported that EDIL3 is overexpressed in several tumor types, including bladder, pancreas, breast, and liver carcinomas, and associates with tumor progression and poor prognosis 16–23 . EDIL3 has been identified as a novel regulator of EMT in hepatocellular carcinoma cells, as the increased expression of EDIL3 by miRNA-137 downregulation triggers ERK and TGF-βactivation via interaction with the integrin α V β 3 ( 20 ). This integrin plays a crucial role in the growth of brain metastasis in breast cancer 24 . Also, EDIL3 has been identified in the extracellular vesicles of breast cancer cells that may be used for early breast cancer detection in the plasma of patients 21–25 . Since EDIL3 is a regulator of EMT and both EDIL3 and EMT play important roles in the progression of cancer and acquisition of resistance to chemotherapy, we studied the expression of EDIL3 and its impact on paclitaxel resistance. Given that the use of paclitaxel is restricted by acquisition of resistance 26 and that pharmacological agents that inhibit the activation of integrins, such as cilengitide, are available, we also examined if the combination of both agents improved the apoptotic response of breast and prostate cancer cells with the aim of designing new therapeutic strategies for the treatment of cancer. Results EMT-related genes are differentially expressed in paclitaxel-sensitive MDA-MB-468 vs. paclitaxel-resistant MDA-MB-468R breast cancer cells The whole-genome expression profiles of paclitaxelsensitive MDA-MB-468 and paclitaxel-resistant MDAMB-468R, were analyzed with the aim of identifying genes that are involved in the acquisition of paclitaxel resistance. Affymetrix Human Gene 1.0 ST Array analyses of mRNA isolated from MDA-MB-468 and MDA-MB-468R cells allowed the identification of 799 genes with differential expression. Out of these genes, 353 were downregulated, whereas 446 were upregulated in MDA-MB-468R vs. MDA-MB-468, including EDIL3,SNAI2/Slug,SPOCK1, and Vimentin, all of which are involved in EMT (Fig. 1a and Supplementary Table S1). The available dataset E-GEOD-12791 contains the whole-genome expression profiles of MDA-MB-231 breast cancer cell line, and its derived paclitaxelresistant RMDA-MB-231 cell line analyzed with the Affymetrix Human Genome U133A Array. In this dataset, out of 337 differentially expressed genes, 159 were downregulated and 178 were upregulated in RMDA-MB231 vs. MDA-MB-231 (Fig. 1b and Supplementary Table S2). EDIL3 and SDC2 were among the upregulated genes in both paclitaxel-resistant MDA-MB-468R and RMDAMB-231 cells, showing a strong significance in the metaanalysis of both datasets (adjusted P<10 −15 ; Fig. 1c and Supplementary Table S3). We validated EDIL3 and SDC2 gene expression by quantitative real-time PCR (qRT-PCR) in paclitaxelsensitive MDA-MB-468, and paclitaxel-resistant MDAMB-468R and MDA-MB-231 cells. These results showed that MDA-MB-231 and MDA-MB-468R cells have significantly higher levels of EDIL3 and SDC2 than MDA-MB468 cells. All these data are in concordance with the results obtained by microarray expression, and indicate that EDIL3 and SDC2 are overexpressed in paclitaxel-resistant MDAMB-231 and MDA-MB-468R, as compared with paclitaxelsensitive MDA-MB-468 cells (Fig. 1d). High levels of EDIL3 expression are associated with the mesenchymal phenotype in paclitaxel-resistant cancer cells First, we studied the protein expression levels of EDIL3 in five breast cancer cell lines, which show differences in paclitaxel sensitivity 27 . The highest levels of EDIL3 were observed in MDA-MB-231 cells, followed by MDA-MB468R, both resistant to paclitaxel; the lowest levels of EDIL3 were observed in SKBR3 cells, followed by MDAMB-468 and BT474 cells, all of them sensitive to paclitaxel (Fig. 2a). Although different protein expression levels of EDIL3 were observed in these breast cancer cell lines, the statistical analysis showed that when compared with MDA-MB-468 cells, only MDA-MB-468R and MDA-MB-231 cells had a significant increase in EDIL3 protein expression (Fig. 2a). We chose these three cell lines to examine the protein expression of epithelial marker E-cadherin and mesenchymal markers, vimentin and Slug. MDA-MB-468 cells had the highest levels of E-cadherin, as well as the lowest levels of vimentin and Slug, indicating that this cell line has an epithelial phenotype. Conversely, MDA-MB231 and MDA-MB-468R cells had the lowest levels of E-cadherin, as well as the highest levels of vimentin and Slug, indicating a mesenchymal phenotype (Fig. 2b). Gasca et al. Cell Death Discovery (2020) 6:86 Page 2 of 14 Official journal of the Cell Death Differentiation Association Fig. 1 Epithelial–mesenchymal transition-related genes are differentially expressed in paclitaxel-sensitive vs. paclitaxel-resistant breast cancer cells. a Heat-map log2 representing the top 50 differentially expressed genes in paclitaxel-sensitive MDA-MB-468 vs. paclitaxel-resistant MDAMB-468R cells. Each column represents a sample and each row represents a gene. Red are upregulated genes and green are downregulated genes. b Heat-map log2 representing the top 50 differentially expressed genes in MDA-MB-231 and paclitaxel-resistant RMDA-MB-231 cells obtained from the dataset E-GEPD-1279. Each column represents a sample and each row represents a gene. Red are upregulated genes and green are downregulated genes. cVenn diagram representing differentially expressed genes from MDA-MB-468 vs. MDA-MB-468R comparison in green, and from MDA-MB231 vs. RMDA-MB-231 comparison in blue. List of 45 common differentially expressed genes is presented. dqRT-PCR analysis of EDIL3 and SDC2 genes in MDA-MB-468, MDA-MB-468R, and MDA-MB-231 breast cancer cells. The quantity of each transcript was divided by the quantity of TBP and EIF2B2 to obtain a normalized value. Data are presented as mean ± SEM (n≥3). *P< 0.05 from Student’sttest; NS not significant. Gasca et al. Cell Death Discovery (2020) 6:86 Page 3 of 14 Official journal of the Cell Death Differentiation Association Fig. 2 (See legend on next page.) Gasca et al. Cell Death Discovery (2020) 6:86 Page 4 of 14 Official journal of the Cell Death Differentiation Association In summary, paclitaxel-resistant breast cancer cells overexpressed EDIL3 and, surprisingly, these cell lines had a mesenchymal phenotype. To evaluate these results in other types of cancer, we selected paclitaxel-resistant PC3 and paclitaxel-sensitive LNCaP prostate cancer cells. The expression of EDIL3 and mesenchymal markers vimentin and Slug were significantly higher in paclitaxelresistant PC3 cells than in paclitaxel-sensitive LNCaP cells, while the expression of epithelial marker E-cadherin was higher in LNCaP than in PC3 cells (Fig. 2c). Again, EDIL3 was overexpressed in paclitaxel-resistant cells, which showed a mesenchymal phenotype. We also performed an immunohistochemical analysis of EDIL3 protein in tumor tissues from 89 breast and 51 prostate cancer patients. Tumor cells showed cytoplasmic immunostaining for this protein. In breast cancer, out of 53 grades I and II breast tumors, 35 (66.0%) expressed low levels of EDIL3, while out of 36 grade III breast tumors, 21 (58.3%) expressed high levels of EDIL3 (Fig. 2d). In prostate cancer, out of 28 low-grade (Gleason ≤7) prostate tumors, 20 (71.4%) expressed low levels of EDIL3, while out of 23 high-grade (Gleason 8–10) prostate tumors, 20 (87%) expressed high levels of EDIL3 (Fig. 2e). Statistical analysis of these results showed a significant positive association between tumor grade and EDIL3 expression in breast and prostate cancers (P=0.03 and P< 0.0001, respectively). In addition, we analyzed RNAseq of 843 patients with breast cancer from the TCGA database 28 , and we observe a statistical significance in the group of patients with high levels of mRNA of EDIL3 that had a shorter overall survival at 10 years (Supplementary Fig. S1). EDIL3 regulates EMT markers through an autocrine or paracrine mechanism in breast cancer cells Our results and others point out that EDIL3 acts as an EMT regulator 25 . Since EDIL3 interacts with integrin α V β 3 and other ligands of this integrin are secreted to the ECM and present an autocrine regulation mechanism, we investigated whether EDIL3 was secreted to the medium by breast cancer cells expressing different levels of EDIL3. We also examined the effect of cilengitide, a cyclic pentapeptide whose chemical structure is based on the RGD sequence, which blocks the interaction of EDIL3 with integrin α V β 3 ( 29 ), on the secretion of EDIL3 and the expression of EMT markers. To choose the optimal concentration of cilengitide, we determined the half inhibitory concentration (IC50) for this agent in MDA-MB-231 cells because this cell line had the highest levels of EDIL3. MDA-MB-231 cells were treated with different concentrations of cilengitide from 10 −4 to 10 2 µM during 72 h, and cell viability was evaluated. The IC50 of cilengitide was calculated as 1.25 ± 0.04 µM in this breast cancer cell line (Fig. 3a), so subsequent experiments were carried out with 1 µM cilengitide. MDA-MB-468, MDA-MB-468R, and MDA-MB-231 cells were treated with dimethyl sulfoxide (DMSO) or 1 µM cilengitide during 48 h, and the concentration of secreted EDIL3 was measured by enzyme-linked immunosorbent assay (ELISA). Our results demonstrated that the three cell lines secreted EDIL3, with the lowest concentration of secreted EDIL3 observed in paclitaxelsensitive MDA-MB-468 cells, and the highest in paclitaxel-resistant MDA-MB-468R and MDA-MB-231 cells, similar to the intracellular EDIL3 expression levels reported in these cells. Interestingly, after cilengitide treatment, the concentration of secreted EDIL3 increased from 1.46 ± 0.12 to 2.76 ± 0.22 ng/ml in MDA-MB-468 cells, from 4.13 ± 0.63 to 6.03 ± 0.93 ng/ml in MDA-MB468R cells, and from 15.73 ± 1.67 to 20.73 ± 2.12 ng/ml in MDA-MB-231 cells (Fig. 3b). We also observed that increased levels of secreted EDIL3 after cilengitide treatment were accompanied by the upregulation of E-cadherin, and the downregulation of intracellular EDIL3 and vimentin in MDA-MB-231 and MDA-MB-468 cells, and Slug in MDA-MB-231 cells (Fig. 3c, d). These results suggest that extracellular levels of EDIL3 and its involvement in EMT regulation could be mediated by an autocrine or paracrine mechanism. To further investigate this hypothesis, we examined the effect of extracellular EDIL3 in MDA-MB-468 cells using EDIL3-enriched conditioned medium (CM) collected from MDA-MB-231 cells. MDA-MB-468 cells growing in (see figure on previous page) Fig. 2 High EDIL3 protein expression is associated with a mesenchymal phenotype in paclitaxel-resistant cancer cells. Western blot analysis of basal expression of EDIL3, and different epithelial and mesenchymal markers are shown. β-actin is shown as a loading control. Histograms show the densitometric analyses of indicated proteins. Data are presented as mean ± SEM (n≥3). aEDIL3 expression in MDA-MB-468, MDA-MB-468R, BT474, SKBR3, and MDA-MB-231 breast cancer cells. bE-cadherin, vimentin, and Slug expression in MDA-MB-468, MDA-MB-468R, and MDA-MB-231 breast cancer cells. cEDIL3, E-cadherin, vimentin, and Slug expression in LNCaP and PC3 prostate cancer cells. d,eRepresentative micrographs of EDIL3 immunohistochemistry in lowand high-grade breast tumors (d), and in lowand high-grade prostate tumors (e). Bars, 75 µm. Histograms show percent of breast (d) or prostate (e) tumors with high EDIL3 expression. Association between EDIL3 and tumor grade was analyzed by Fisher’s exact test. *P< 0.05 from Student’sttest; NS not significant. Gasca et al. Cell Death Discovery (2020) 6:86 Page 5 of 14 Official journal of the Cell Death Differentiation Association CM showed higher expression of EDIL3, vimentin, and Slug, and lower expression of E-cadherin in comparison with MDA-MB-468 growing in unconditioned medium (UM), and this trend reverted when MDA-MB-468 growing in CM were treated with cilengitide (Fig. 3d). Furthermore, an immunohistochemical analysis of Ecadherin was performed to confirm this hypothesis (Supplementary Fig. S2). Together these results confirm that extracellular EDIL3 regulates EMT through an autocrine or paracrine mechanism, involving its interaction with integrin α V β 3 . EDIL3 gene silencing increases paclitaxel-induced apoptosis and reverts EMT in paclitaxel-resistant cancer cells Because the paclitaxel-resistant MDA-MB-231 and PC3 cell lines presented the highest levels of EDIL3 and a mesenchymal phenotype, they were selected to gain insight into the involvement of EDIL3 in paclitaxel resistance and EMT. For that, EDIL3 expression was silenced using a specific pool of small interfering RNA (siRNA), and breast and prostate cancer cells were subsequently treated with 1 or 2.5 µM paclitaxel during 48 h, Fig. 3 EDIL3 regulates epithelial–mesenchymal transition through an autocrine or paracrine mechanism in breast cancer cells. a IC50 curve for cilengitide in MDA-MB-231 breast cancer cell line. Data are presented as mean ± SEM. bHistogram shows the concentration of EDIL3 protein in the medium determined by ELISA in MDA-MB-468, MDA-MB-468R, and MDA-MB-231 breast cancer cells treated with DMSO or 1 µM cilengitide during 48 h. cWestern blot analysis of EDIL3, E-cadherin, vimentin, and Slug in MDA-MB-231 cells treated with DMSO and 1 µM cilengitide during 48 h. β-actin is shown as a loading control. Histograms show the densitometric analyses of indicated proteins. Data are presented as mean ± SEM (n≥3). dWestern blot analysis of EDIL3, E-cadherin, vimentin, and Slug in MDA-MB-468 cells cultured in unconditioned medium (UM) or EDIL3enriched conditioned medium collected from MDA-MB-231 cells (CM), and treated with DMSO and 1 µM cilengitide during 48 h. β-actin is shown as a loading control. Histograms show the densitometric analyses of indicated proteins. Data are presented as mean ± SEM (n≥3). *P< 0.05 from Student’sttest; NS not significant. Gasca et al. Cell Death Discovery (2020) 6:86 Page 6 of 14 Official journal of the Cell Death Differentiation Association respectively. EDIL3-silenced MDA-MB-231 cells showed increased PARP cleavage and caspase-3 activation after paclitaxel treatment, as compared with siRNA control cells (Fig. 4a). We also confirm the role of EDIL3 in the paclitaxel resistance with a long-term viability assay in this cell line (Supplementary Fig. S3a). With respect to EMT markers, EDIL3-silenced MDA-MB-231 cells showed increased expression of E-cadherin, while the expression of vimentin and Slug were decreased as compared with siRNA control cells (Fig. 4a). Although paclitaxel treatment induced the upregulation of E-cadherin levels, especially in EDIL3-silenced MDA-MB-231 cells, the levels of vimentin or Slug were not significantly affected by paclitaxel treatment (Fig. 4a). In the case of PC3 cells, the induction of apoptosis was more potent in EDIL3-silenced PC3 cells as evidenced by the increase in cleaved PARP and active caspase-3 after paclitaxel treatment, as compared with siRNA control cells (Fig. 4b). Regarding the EMT markers, EDIL3 gene silencing induced the downregulation of vimentin and Slug, as well as the upregulation of E-cadherin. Although an increase in E-cadherin and vimentin was observed after paclitaxel treatment, statistically significant differences were not observed between paclitaxel-treated siRNA Fig. 4 EDIL3 gene silencing increases paclitaxel-induced apoptosis and reverts epithelial–mesenchymal transition in paclitaxel-resistant cancer cells. a MDA-MB-231 and bPC3 cells were silenced for EDIL3, and treated with DMSO and 1 or 2.5 µM paclitaxel, respectively. Paclitaxelinduced apoptosis was assessed by western blot analysis of cleaved PARP and active caspase-3. Modulation of EDIL3, E-cadherin, vimentin, and Slug was studied by western blot analysis, using β-actin as a loading control. Histograms show the densitometric analyses of indicated proteins. Data are presented as mean ± SEM (n≥3). *P< 0.05 from Student’sttest; NS not significant. Gasca et al. Cell Death Discovery (2020) 6:86 Page 7 of 14 Official journal of the Cell Death Differentiation Association control PC3 cells and paclitaxel-treated EDIL3-silenced PC3 cells (Fig. 4b). The invasion ability of PC3 cells was tested in a Matrigel assay, in which was confirmed that the downregulation of endogenous EDIL3 expression decreases the invasive potential of PC3 cells (Supplementary Fig. S3b). Extracellular EDIL3 or EDIL3 overexpression favors EMT and reduces paclitaxel sensitivity in cancer cells As paclitaxel-sensitive MDA-MB-468 and LNCaP cells presented the lowest levels of EDIL3 and an epithelial phenotype, they were selected to study the effect of extracellular EDIL3 or EDIL3 overexpression on the response to paclitaxel and in the regulation of EMT. The effect of extracellular EDIL3 was examined using CM collected from MDA-MB-231 cells and used to culture MDA-MB-468 cells, subsequently treated with DMSO or 1 µM paclitaxel during 48 h. MDA-MB-468 cells growing in CM showed more resistance to paclitaxel-induced apoptosis than MDA-MB-468 cells cultured in UM, as indicated by the lower levels of cleaved PARP and caspase-3 (Fig. 5a). This was also verified by annexin V Fig. 5 Extracellular EDIL3 and EDIL3 overexpression promotes epithelial–mesenchymal transition and paclitaxel resistance in cancer cells. aMDA-MB-468 breast cancer cells were cultured in unconditioned medium (UM) or EDIL3-enriched conditioned medium collected from MDA-MB231 cells (CM), and treated with DMSO and 1 µM paclitaxel during 48 h. bLNCaP prostate cancer cells were transiently transfected with pCMV6-XL4EDIL3 or with empty vector, and treated with DMSO or 2.5 µM paclitaxel during 48 h. Paclitaxel-induced cleavage of PARP and caspase-3, as well as paclitaxel-induced modulation of EDIL3, E-cadherin, vimentin, and Slug were analyzed by western blot using β-actin as a loading control. Histograms represent the densitometric analyses of indicated proteins. Data are presented as mean ± SEM (n≥3). *P< 0.05 from Student’sttest; NS, not significant. Gasca et al. Cell Death Discovery (2020) 6:86 Page 8 of 14 Official journal of the Cell Death Differentiation Association binding assays (Supplementary Fig. S4). Furthermore, additional experiments using CM from EDIL3 and control siRNA-treated MD-MB-231 cells showed that paclitaxel sensitivity was restored in MDA-MB-468 cells grown in siRNA EDIL3 CM, both by western blot and annexin V binding assays (Supplementary Figs. S5a, b and S6). Together these results further confirm that extracellular EDIL3 regulates paclitaxel response in breast cancer cells. Also, cells cultured in CM showed a more mesenchymal phenotype than cells cultured in UM. Although no effect on Slug expression was observed after paclitaxel treatment, the downregulation of EDIL3 and E-cadherin, as Fig. 6 Cilengitide sensitizes paclitaxel-resistant cancer cells to paclitaxel-induced apoptosis and reverts mesenchymal transition. a MDAMB-231 and bPC3 cells were treated with DMSO, 1 µM cilengitide, 1 or 2.5 µM paclitaxel, and 1 µM cilengitide plus 1 or 2.5 µM paclitaxel, respectively. The induction of apoptosis was studied through the cleavage of PARP and caspase-3 by western blot, as well as the expression of indicated epithelial–mesenchymal transition markers. β-actin was used as a loading control. Histograms show the densitometric analyses of indicated proteins. Data are presented as mean ± SEM (n≥3). *P< 0.05 from Student’sttest; NS not significant. Gasca et al. Cell Death Discovery (2020) 6:86 Page 9 of 14 Official journal of the Cell Death Differentiation Association