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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 αvβ3 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, αvβ3, 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, αvβ3 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 αvβ3 as a marker/driver of carcinoma stemness but also reveal a therapeutic strategy to sensitize such tumours to RTK inhibition.

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Figure 1: Integrin β3 expression drives a TIC phenotype.
Figure 2: Integrin β3 drives RTK inhibitor resistance.
Figure 3: Acquired resistance to EGFR inhibition selects for a β 3 + cell population with TIC properties.
Figure 4: Integrin-β3–KRAS complex is critical for integrin-β3-mediated tumour-initiating phenotype and EGFR inhibitor resistance.
Figure 5: Galectin-3 is essential for the integrin-β3–KRAS complex.
Figure 6: RalB is a key modulator of integrin-β3-mediated tumour-initiating phenotype and EGFR inhibitor resistance.
Figure 7: TBK1 and c-Rel inhibition overcome β3-mediated stemness and EGFR inhibitor resistance.
Figure 8: Model depicting the proposed integrin-αvβ3-mediated EGFR TKI resistance mechanism.

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Acknowledgements

We thank D. Shields, E. Murphy, L. Acevedo, S. Advani, M. Huang, I. Tancioni, B. Walsh and A. Larange for helpful discussions. We thank D. Young for his advice and the technical support with the FACS sorter. We thank the Moores Cancer Center Biorepository and H. Howard for their help with the PDXs. We thank J. Camonis for providing RalB constructs. We also thank C. Mirsaidi from Molecular Response LLC for providing the PDXact human lung cancer patient-derived xenograft models used here. D.A.C. was supported by US National Institutes of Health grants CA45726, CA168692, HL57900 and R3750286. A.M.L. was supported by NIH CA155620. S.K. was supported by the National Cancer Institute of the National Institutes of Health under award number T32CA121938. L.S. was supported by the Association pour la Recherche sur le Cancer (ARC) and La Fondation Philippe.

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Authors and Affiliations

Authors

Contributions

L.S. designed and carried out experiments, interpreted data, and wrote the paper; S.K., A.F., J.L., M.Y., M.F.C. and K.C.E. carried out experiments; S.K. provided the lung biopsies. A.M.L. provided the pancreatic PDXs. T.C. and J.V.H provided the H441 model. T.C., J.V.H., L.D., J.W., I.I.W. and J.V.H. provided the BATTLE study. S.M.W., J.S.D., A.M., H.H. and S.A. gave conceptual advice, and S.M.W. and D.A.C. designed experiments, interpreted data and wrote the paper.

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Correspondence to David A. Cheresh.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Integrin β3 drives a tumour initiating phenotype in histologically distinct tumour cell types.

(a) Limiting dilution experiments determining tumour-initiation efficiency shown in Fig. 1a. (b) Tumour incidence of integrin β 3 - and integrin β 3 + subpopulations of cells from 1 pancreatic cancer patient-derived xenograft. Cells were tested for tumour initiation in NOD/SCIDIl2r−/−mice. n = 4 injections in 2 mice per group. (c) Phase-contrast images of tumour spheres seeded with β 3 + and β 3 - cells from a lung NSCLC patient tumour (patient #199600628). Scale bar, 100 μm. (d) Immunoblots showing integrin β3 ectopic expression or knockdown efficiency in cells used in this study. (ef) Limiting dilution experiments determining tumour-initiation efficiency (% tumour induction) shown in Fig. 1d (e) and e (f). (g) Phase contrast images of self-renewal secondary tumour spheres of A549, PANC-1, MDA-MB-231-Lm2 and FG expressing or lacking integrin β3. Scale bar, 100 μm. P value was estimated by χ2 test in a,e,f. Uncropped western-blots are provided in Supplementary Figure 7.

Supplementary Figure 2 Integrin β3 promote RTK inhibitor resistance independently of its RGD binding domain.

(a) Representative photographs of crystal violet-stained tumour spheres of β 3 - and β 3 + cells after vehicle, erlotinib, lapatinib and linsitinib treatment. (b) Effect of integrin β3 knockdown (sh3) on erlotinib response in lung cancer cells A549 and H1650 and in breast cancer cells MDA-MB-231-Lm2 grown in 3D. The bar graph represents the mean ± s.d. for n = 3 independent experiments. (c) Immunoblot showing integrin 3 knockdown efficiency in H1650 cells. (d) Immunoblot showing erlotinib treatment efficiency in tumours shown in Fig. 2e. (e) Erlotinib and Lapatinib IC50 in a panel of human carcinoma cell lines treated with erlotinib (lung and pancreas) or lapatinib (breast) in 3D culture Date are representative of 2 independent experiments. IC50 are calculated using Prism GraphPad software. (f) Effect of cilengitide treatment on tumour-sphere formation and erlotinib resistance in FGβ3 and PANC-1 cells. The bar graph represents the mean of 3 wells per group. Data are representative of 2 independent experiments. (g) Effect of ectopic expression of β3 wild-type (FGβ3) or the β3 D119A (FGD119A) ligand binding domain mutant on erlotinib response. Data are mean for 3 wells per group. Data are representative of 2 independent experiments. Immunoblot showing transfection efficiency of vector control, integrin β3 wild-type and integrin β3 D119A. P value was estimated by Student’s t-test in b. *P < 0.05, **P < 0.01. Uncropped western-blots are provided in Supplementary Figure 7, and original data for b,f,g are provided in the Statistical Source data (Supplementary Table 3).

Supplementary Figure 3 Enrichment of integrin β 3 + cells during acquired resistance to erlotinib.

(a) Immunoblot of integrin β3 in tumour lysates from Fig. 3a. (b) Representative Immunohistochemistry staining of integrin β3 in mouse orthotopic lung tumours after systemic treatment with vehicle or erlotinib (58 days) shown in Fig. 3c. Scale bar, 50 μm. (c) Representative immunofluorescent staining of integrin αvβ3 in pancreatic human xenografts treated 4 weeks with vehicle or erlotinib shown in Fig. 3d. Scale bar, 40 μm. (d) Phase contrast images of self-renewal tumour spheres of HCC827 vehicle treated, erlotinib treated unsorted, β 3 - and β 3 + sorted population. Scale bar, 100 μm. (e) Limiting dilution experiments determining tumour-initiation efficiency (% tumour induction) shown in Fig. 3f. (f) Relative mRNA expression of ALDH1 in HCC827 vehicle-treated tumours (n = 5 tumours) or erlotinib-treated tumours (n = 7 tumours) from Fig. 3a. mean ± SEM. (g) Effect of integrin β3 knockdown on ALDH1 expression in A549 in 3D culture. Results are represented as fold change in mRNA expression in A549 shβ3 versus control. Data are mean ± s.d. n = 3 independent experiments. P value was estimated by χ2 test in e. Students t-test in f,g. *P < 0.05, **P < 0.01. Uncropped western-blots are provided in Supplementary Figure 7. Original data for g are provided in the Statistical source data (Supplementary Table 3).

Supplementary Figure 4 KRAS is required for integrin β3-mediated erlotinib resistance.

(a) Confocal microscopy images of FGβ3 shCTRL and FGβ3 shKRAS cells grown in 3D and stained for KRAS. Immunoblot showing KRAS knockdown efficiency in those cells. Scale bar, 10 μm. (b) Confocal microscopy images show immunostaining for Integrin β3 (green), KRAS (red) and DNA (TOPRO-3, blue) for PANC-1, A549 and HCC827 after acquired resistance to erlotinib (HCC827 ER) grown in 3D. Scale bar, 10 μm. Data are representative of three independent experiments. (c) Quantified percentage of cells with colocalization of Integrin β3 and KRAS shown in(b). Data shown represent mean ± SEM for n (total fields counted over 3 independent experiments) = 10 fields per group. (d) Immunoblots showing KRAS knockdown efficiency in cells used in Fig. 4. (e) Representative photographs of crystal violet-stained tumour spheres of FG and FGβ3 cells expressing non-silencing shCTRL or KRAS specific shRNA and treated with vehicle or erlotinib. (f) Effect of KRAS knockdown on erlotinib and serum deprivation response measured by CellTiterGLO cell viability assay for FG cells grown in 3D and treated with 1 μM erlotinib or in absence of serum. Data are expressed in relative Luciferase Units (RLU). The bar graph represents the mean ± s.d. for n = 3 independent experiments. (g) Confocal microscopy images of FGβ3 cells grown in 3D and stained for integrin αvβ3 (red) and KRAS2A or KRAS2B. Scale bar, 10 μm. Data are representative of three independent experiments. (h) Immunoblots showing Galectin-3 knockdown efficiency in cells used in Fig. 5. P value was estimated by Students t-test in f. Uncropped western-blots are provided in Supplementary Figure 7. Original data for c,f are provided in the Statistical source data (Supplementary Table 3).

Supplementary Figure 5 RalB is required for integrin β3-mediated erlotinib resistance and stemness.

(a) Effect of ERK, AKT, RalA and RalB knockdown on erlotinib response for FG and FGβ3 cells. Immunoblots showing ERK, AKT, RalA and RalB knockdown efficiency. The bar graph represents mean for 3 wells per groups. Data are representative experiment of 2 independent experiments. (b) Immunoblots showing RalB knockdown efficiency in cells used in Fig. 6. (c) Limiting dilution experiments determining tumour-initiation efficiency shown in Fig. 6c. (d) Phase contrast images of self-renewal tumour spheres of FGβ3 expressing non-silencing shRNA CTRL or RalB specific shRNA. Scale bar, 100 μm. (e) Effect of expression of RalB wild-type (RALB WT) or constitutively active mutant (RalB G23V) on erlotinib response of FG cells. The bar graph represents mean for 4 wells per groups. Data are representative experiment of 2 independent experiments. Immunoblot showing RalB WT and G23V transfection efficiency. Representative photographs of crystal violet-stained tumour spheres of FG transfected with CTRL, RalB WT or RalB G23V and treated with Vehicle or erlotinib 0.5 μM. P value was estimated by χ2 test in c. Uncropped western-blots are provided in Supplementary Figure 7. Original data for a,e are provided in the Statistical source data (Supplementary Table 3).

Supplementary Figure 6 TBK1 and c-Rel inhibition overcome β3-mediated erlotinib resistance.

(a) Immunoblots showing TBK1 and c-Rel knockdown efficiency in cells used in this study. (b) Phase contrast images of self-renewal tumour spheres of FGβ3 expressing non-silencing shCTRL or TBK1 specific shRNA. Scale bar, 100 μm. (c) Effect of TBK1 and c-Rel knockdown measured by CellTiterGLO cell viability assay for FG cells. Cells were grown in 3D. The bar graph represents mean for 2 wells per group. Data are representative experiment of 2 independent experiments. (d) Confocal microscopy images of c-Rel (red) and DNA (TOPRO-3, blue) in FGβ3 cells treated with vehicle, or bortezomib 20 nM. Scale bar, 50 μm. (e) Confocal microscopy images of cleaved caspase 3 (red) and DNA (TOPRO-3, blue) in FGβ3 tumour biopsies from xenografts tumours used in Fig. 7g treated with vehicle, erlotinib, bortezomib or bortezomib and erlotinib in combo. Scale bar, 100 μm. Uncropped western-blots are provided in Supplementary Figure 7. Original data for c are provided in the Statistical source data (Supplementary Table 3).

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Seguin, L., Kato, S., Franovic, A. et al. An integrin β3–KRAS–RalB complex drives tumour stemness and resistance to EGFR inhibition. Nat Cell Biol 16, 457–468 (2014). https://doi.org/10.1038/ncb2953

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