An integrin β₃-KRAS-RalB complex drives tumour stemness and resistance to EGFR inhibition

Abstract

Tumour cells, with stem-like properties, are highly aggressive and often show drug resistance. Here, we reveal that integrin α(v)β₃ serves as a marker of breast, lung and pancreatic carcinomas with stem-like properties that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib. This was observed in vitro and in mice bearing patient-derived tumour xenografts or in clinical specimens from lung cancer patients who had progressed on erlotinib. Mechanistically, α(v)β₃, in the unliganded state, recruits KRAS and RalB to the tumour cell plasma membrane, leading to the activation of TBK1 and NF-κB. In fact, α(v)β₃ expression and the resulting KRAS-RalB-NF-κB pathway were both necessary and sufficient for tumour initiation, anchorage independence, self-renewal and erlotinib resistance. Pharmacological targeting of this pathway with bortezomib reversed both tumour stemness and erlotinib resistance. These findings not only identify α(v)β₃ as a marker/driver of carcinoma stemness but also reveal a therapeutic strategy to sensitize such tumours to RTK inhibition.

Trial registration: ClinicalTrials.gov NCT00409968.

Figure 1. Integrin β3 expression drives a tumor-initiating cell phenotype

(a) Frequency of tumor-initiating cells (TIC) in unsorted, integrin β3⁻ and integrin β3⁺ subpopulations of cells from 3 NSCLC patients-derived xenografts. Cells were tested for tumor initiation in NOD/SCID Il2rγ^−/− (NSG) mice. The n number of injection per condition for each patient are shown in Supplementary Figure 1a. The frequency of tumor-initiating cells per 10,000 cells was calculated using the ELDA extreme limiting dilution software. (b) Quantification of tumorspheres formed by the unsorted, the β3⁻ and the β3⁺ populations from 3 NSCLC patients. n=3 independent experiments (3 technical replicates per experiment); mean ± SD.(c) Histological analysis of patient primary and β3⁺ subpopulation tumors. Tumors were stained for Integrin β3. Scale bar, 100 μm.(d)Frequency of tumor-initiating cells for A549 cells expressing shCTRL or shβ3 in NSG and nude mice. The n number of injection per condition for A549 shCTRL and A549 shβ3 are shown in Supplementary Figure 1d.(e)Frequency of tumor-initiating cells for FG cells expressing control vector or integrin β3 (FGβ3) in nude mice. The n number of injection per condition for FG and FGβ3 are shown in Supplementary Figure 1e.(f) Self-renewal capacity of A549, PANC-1 and MDA-MB-231 Lm2 cells expressing shCTRL or shβ3 measured by quantifying the number of primary and secondary tumorspheres. n= 3 independent experiments for PANC-1 and A549 (3 technical replicates per experiment). For MDA-MB-231 Lm2, n= 3 technical replicates of a representative experiment. mean ± SD. (g)Self-renewal capacity of FG expressing control vector (+ CTRL) or integrin β3 (+β3), measured by quantifying the number of primary and secondary tumorspheres. n= 3 independent experiments (3 technical replicates per experiment); mean ± SD. P value was estimated by Student’s t-test in b,f,g; χ² test in a,d,e.*P< 0.05, **P< 0.01, ***P< 0.001. Original data for b,f,g are provided in the Statistical Source data (Supplementary Table 3).

Figure 2. Integrin β3 drives RTK inhibitor resistance

(a)Effect of serum deprivation on FG and FGβ3 cells measured by CellTiterGLO cell viability assay. Cells were grown in 3D in media with media containing 10% serum or 0% serum. Data are expressed in relative Luciferase Units (RLU). n= 3 independent experiments (2 technical replicates per experiment for CTRL and 3 technical replicates for +β3); mean ± SD.(b)Effect of integrin β3 expression (FG and FGβ3) on drug treatment response in pancreatic cancer cells. Cells in 3D culture were treated with a dose response of erlotinib, lapatinib, linsitinib, gemcitabine and cisplatin. IC50 are calculated using Graph Pad prism software. Data are representative of 2 independent experiments.(c–d) Effect of erlotinib treatment on lung (c) and pancreatic (d) cells expressing or lacking integrin β3 measured by CellTiterGLO cell viability assay. Cells were grown in 3D in media with 1μM of erlotinib. Data are expressed in relative Luciferase Units (RLU). n= 3 independent experiments (3 technical replicates per experiment); mean ± SD. (e)Effect of integrin β3 knockdown on erlotinib resistance in vivo, A549 shCTRL and A549 shβ3 (n= 8 mice per treatment group) were treated with erlotinib (25 mg/kg/day) or vehicle during 16 days. Tumor volumes are expressed as mean ± SEM. (f) Orthotopic FG and FGβ3 tumors (>1000 mm³; n= 7 mice for FG and n= 8 mice FGβ3 vehicle and n= 5 mice for each treatment group) were treated for 30 days with vehicle or erlotinib. Results are expressed as % tumor weight compared to vehicle control. mean ± SEM. P value was estimated by Student’s t-test in a,c,d,e,f.*P< 0.05, **P< 0.01, ***P< 0.001. Original data for a,b,c,d are provided in the Statistical Source data (Supplementary Table 3).

Figure 3. Acquired resistance to EGFR inhibition selects for a β3+ cell population with tumor-initiating cell properties

(a) Effect of erlotinib treatment on HCC827 xenograft tumors. HCC827 cells were treated with vehicle control or erlotinib (25 mg/kg/day) until acquired resistance. Tumor dimensions are reported as the fold change relative to size of the same tumor on Day 1. n= 8 mice per group. Data are mean ± SEM. (b) Relative mRNA expression of integrin β3 (ITGβ3) in HCC827 vehicle-treated tumors (n= 5 tumors) or erlotinib-treated tumors (n= 7 tumors) from (a). Data are mean ± SEM.(c) Quantification of Immunohistochemistry staining of integrin β3 in mouse orthotopic lung H441 tumors treated with vehicle (n= 8 tumors) or erlotinib (n= 7 tumors) was scored (scale 0 to 3).(d) Quantification of integrin αvβ3 expression in pancreatic human FG xenografts treated 4 weeks with vehicle (n=3 tumors) or erlotinib (n=4 tumors). Integrin αvβ3 expression was quantified as ratio of integrin αvβ3 pixel area over nuclei pixel area using Metamorph software. Data are mean. (e) Self-renewal capacity of HCC827 vehicle-treated (vehicle) and erlotinib-treated tumors (erlotinib resistant unsorted) from (a). The HCC827 erlotinib-treated tumors have been sorted in two groups: the integrin β3⁻ and the integrin β3⁺ population. n= 3 independent experiments (3 technical replicates per experiment); mean ± SD. (f) Frequency of tumor-initiating cells for HCC827 vehicle-treated (vehicle), erlotinib-treated (erlotinib resistant unsorted), erlotinib-treated β3⁻ population and erlotinib-treated β3⁺ population. The n number of injection per condition for FG and FGβ3 are shown in Supplementary Figure 3e (g) Histological analysis of primary HCC827 erlotinib resistant xenografts and Integrin β3⁺ and Integrin β3⁻ subpopulations. Tumors were stained for H&E and Integrin β3. Scale bar, 50 μm. (h) Box plot comparing integrin β3 (ITGβ3) gene expression in human lung cancer biopsies from patients from the BATTLE Study who were previously treated with an EGFR TKI and progressed (n=31 patients), versus patients who were EGFR TKI naïve (n=43 patients). The box shows the median and the interquartile range. The wiskers show the minimum and maximum. (i) Immunohistochemical analysis of integrin β3 expression in paired human primary lung cancer biopsies obtained before and after erlotinib resistance. Scale bar, 100 μm. P value was estimated by Student’s t-test in b,c,d,e,h; χ² test in f.*P< 0.05, **P< 0.01. ***P< 0.001. Original data for d,e are provided in the Statistical Source data (Supplementary Table 3).

Figure 4. Integrin β3/KRAS complex is critical for integrin β3-mediated tumor-initiating phenotype and EGFR inhibitor resistance

(a) Confocal microscopy images of FGβ3 cells grown in 3D and stained for integrin αvβ3 (green) and RAS family members (red). Scale bar, 10 μm. Data are representative of three independent experiments. (b)Quantified percentage of cells with colocalization of Integrin β3 and RAS in (a). Data shown represent mean ± SEM. n= 11 fields for KRAS and RRAS and 10 fields for HRAS and NRAS.(c) Immunoblot analysis of KRAS immunoprecipitates from FG and FGβ3 cells. Data are representative of three independent experiments. (d) Effect of KRAS knockdown on tumorspheres formation in lung and pancreatic cancer cells expressing or lacking integrin β3. n= 3 independent experiments (3 technical replicates per experiment); mean ± SD.(e) Effect of KRAS knockdown on β3-mediated erlotinib resistance measured by CellTiterGLO cell viability assay for FGβ3, PANC-1 and A549. Cells were grown in 3D in media and treated with 1μM of erlotinib. Data are expressed in relative Luciferase Units (RLU). n= 3 independent experiments (2 technical replicates per experiment); mean ± SD. (f) Effect of KRAS knockdown on β3-mediated survival under serum deprivation measured by CellTiterGLO cell viability assay for FGβ3, PANC-1 and A549. Cells were grown in 3D in media containing 10% or 0% serum. Data are expressed in relative Luciferase Units (RLU). n= 3 independent experiments (2 technical replicates per experiment); mean ± SD. (g) Self-renewal capacity of FGβ3 cells expressing non-target shRNA control (shCTRL) or KRAS-specific shRNA (shKRAS) measured by quantifying the number of primary and secondary tumorspheres. n= 3 wells per group. mean ± SD. Data are representative of 2 independent experiments. Phase contrast images of self-renewal tumorspheres of FGβ3 cells expressing non-silencing shRNA CTRL or specific KRAS shRNA. Scale bar, 100 μm. P value was estimated by Student’s t-test in b,d,e,f,g.*P< 0.05, **P< 0.01, ***P< 0.001. Uncropped western-blots are provided in Supplementary Figure 7 and original data for b,d,e,f,g are provided in the Statistical Source data (Supplementary Table 3).

Figure 5. Galectin-3 is essential for Integrin β3/KRAS complex

(a) Confocal microscopy images show immunostaining for integrin β3 (green), KRAS (red) and DNA (TOPRO-3, blue) for PANC-1 cells expressing non-target shRNA control (shCTRL) or Galectin-3-specific shRNA (shGal-3) grown in 3D. Scale bar, 10 μm. Data are representative of three independent experiments. (b) Immunoblot analysis of KRAS immunoprecipitates from PANC-1 cells expressing non-target shRNA control (shCTRL) or Galectin-3-specific shRNA (shGal-3). Data are representative of 3 independent experiments.(c) Effect Galectin-3 knockdown on β3-mediated erlotinib resistance in FGβ3 cells. n= 3 independent experiments (3 technical replicates per experiment); mean ± SD.(d) Effect of Galectin-3 knockdown on erlotinib response measured by CellTiterGLO cell viability assay for FG, and FGβ3. Data are expressed in relative Luciferase Units (RLU). Cells were grown in 3D and treated with 1μM of erlotinib. n=3 independent experiments (2 technical replicates per experiment); mean ± SD. (e) Self-renewal capacity of PANC-1 cells expressing non-target shRNA control (shCTRL) or Galectin-3-specific shRNA (shGal-3) measured by quantifying the number of primary and secondary tumorspheres. n=3 wells per group; mean ± SD. Data are representative of 2 independent experiments. Phase contrast images of self-renewal tumorspheres of PANC-1 cells expressing non-silencing shRNA CTRL or Galectin-3 specific shRNA. Scale bar, 100 μm. P value was estimated by Student’s t-test in c,d,e.*P< 0.05, **P< 0.01, **P< 0.01 Uncropped western-blots are provided in Supplementary Figure 7 and original data for c,d,e are provided in the Statistical Source data (Supplementary Table 3).

Figure 6. RalB is a key modulator of integrin β3-mediated tumor-initiating phenotype and EGFR inhibitor resistance

(a) Effect of RalB knockdown on β3-mediated anchorage independence. n= 3 independent experiments (3 technical replicates per experiment); mean ± SD. (b) Self-renewal capacity of FGβ3 expressing shCTRL or shRalB measured by quantifying the number of primary and secondary tumorspheres. n= 3 independent experiments (3 technical replicates per experiment); mean ± SD. (c) Limiting dilution in vivo determining the frequency of tumor-initiating cells for FGβ3shCTRL and FGβ3 shRalB. The n number of injection per condition for FG and FGβ3 are shown in Supplementary Figure 5c.(d)Effect of RalB knockdown on β3-mediated erlotinib resistance measured by tumorspheres formation. n= 3 independent experiments (3 technical replicates/experiment); mean ± SD.(e–f)Effect of RalB knockdown on β3-mediated erlotinib (e) and nutrient deprivation (f) resistance measured by CellTiterGLO cell viability assay. n= 3 independent experiments (2 technical replicates per experiment); mean ± SD. (g) Effect of RalB knockdown on β3-mediated erlotinib resistance of human pancreatic (FGβ3) orthotopic tumor xenografts. Established tumors expressing shCTRL or shRalB (>1000 mm³) were randomized and treated for 10 days with vehicle or erlotinib (50 mg/kg/day). Results are expressed as tumor weight± SEM. n=6 mice for FGβ3 shCTRL vehicle and erlotinib treated groups and n=11 mice for FGβ3 shRalB vehicle treated, and n=13 mice for FGβ3 shRalB erlotinib treated groups. (h)Left, RalA and RalB activities were determined in cells grown in suspension using a GST-RalBP1-RBD immunoprecipitation assay. Data are representative of three independent experiments. Right, Effect of KRAS knockdown on RalB activity. Data are representative of three independent experiments.(i) Confocal microscopy images of integrin αvβ3 (green), RalB (red) and DNA (TOPRO-3, blue) in tumor biopsies from pancreatic cancer patients. Scale bar, 10 μm.(j) Left, Immunoblots showing expression of indicated proteins in representative FG and FGβ3 tumor xenografts from Figure 2f analyzed at treatment day 30. Right, Immunoblots showing expression of indicated proteins in representative FGβ3 shCTRL and FGβ3 shRalB tumor xenografts from (i) analyzed at treatment day 10. P value was estimated by Student’s t-test in a,b,d,e,f,g; χ² test in c.*P< 0.05, **P< 0.01, ***P< 0.001. Uncropped western-blots are provided in Supplementary Figure 7 and original data for a,b,d,e,f, are provided in the Statistical Source data (Supplementary Table 3).

Figure 7. TBK1 and c-Rel inhibition overcome β3-mediated stemness and EGFR inhibitor resistance

(a) Effect of TBK1 knockdown on PANC-1 self-renewal capacity. n=3 wells per group; mean ± SD. Data are representative of 2 independent experiments. (b) Effect of TBK1 knockdown on erlotinib resistance in FGβ3 measured by tumorspheres formation. n=3 wells per group; mean ± SD. Data are representative of 2 independent experiments.(c–d)Effect of TBK1 and c-Rel knockdown on (c)β3-mediated erlotinib resistance and (d) β3-mediated serum deprivation survival measured by CellTiterGLO cell viability assay for FGβ3 cells. Cells were grown in 3D in and treated with 1 μM of erlotinib (c) or with 0% serum (d). Data are expressed in relative Luciferase Units (RLU). n= 3 independent experiments (2 technical replicates/experiment); mean ± SD.(e–f) Effect of bortezomib treatment on erlotinib resistance measured by (e) tumorspheres formation and (f) CellTiterGLO cell viability assay for FGβ3 cells. Cells were grown in 3D in and treated with 0.5/1μM of erlotinib and 4/20 nM of bortezomib. Data are expressed in relative Luciferase Units (RLU). n=3 wells per group; mean ± SD Data are representative of 2 independent experiments.(g)Mice bearing subcutaneous β3-positive tumors (FGβ3) were treated with vehicle, erlotinib (25 mg/kg/day) or bortezomib (0.25 mg/kg) alone or in combination. Tumor dimensions are reported as the fold change relative to size of the same tumor on Day 1. mean ± SEM. n= 8 mice for vehicle treated, bortezomib treated and erlotinib treated group and n=13 mice for combo treated group. (h) Mice bearing subcutaneous β3-negative HCC827 tumors were treated with erlotinib (25 mg/kg/day) or erlotinib (25mg/kg/day) and bortezomib (0.25 mg/kg) until erlotinib resistance. Tumor volumes are reported. Mean ± SEM. n= 10 mice per group. (i) Immunohistochemical analysis of integrin β3 expression in HCC827 tumors treated 90 days with erlotinib or erlotinib and bortezomib from (h). Scale bar, 50 μm. P value was estimated by Student’s t-test in a,b,c,d,e,f; one way ANOVA test in g,h.*P< 0.05, **P< 0.01. The original data for a,b,c,d,e,f are provided in the Statistical Source data (Supplementary Table 3).

Figure 8. Model Depicting the Proposed Integrin αvβ3-mediated EGFR TKI resistance mechanism

(a–b) During EGFR TKI treatment, a β3-positive cancer stem cell population is selected. Integrin β3 interacts with KRAS via Galectin-3 to promote RalB activation. RalB subsequently activates TBK1 resulting in the activation of the NFkB pathway and thereby promoting cell survival. Importantly, ligation of integrin is not required for this signaling cascade. As demonstrated the inhibition of this non-canonical pathway sensitizes β3-positive tumor cells to EGFR TKI. Targeting this pathway genetically or pharmacologically was able to reverse cancer stemness and drug resistance.