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. 2019 Feb 4;216(2):450-465.
doi: 10.1084/jem.20180742. Epub 2019 Jan 14.

LUBAC determines chemotherapy resistance in squamous cell lung cancer (VSports手机版)

E Josue Ruiz  1 , Markus E Diefenbacher  1 , Jessica K Nelson  1 , Rocio Sancho  1 , Fabio Pucci  1 , Atanu Chakraborty  1 , Paula Moreno  2   3 , Alessandro Annibaldi  4 , Gianmaria Liccardi  4 , Vesela Encheva  5 , Richard Mitter  6 , Mathias Rosenfeldt  7 , Ambrosius P Snijders  5 , Pascal Meier  4 , Marco A Calzado  2   8 , Axel Behrens  9   10
Affiliations

LUBAC determines chemotherapy resistance in squamous cell lung cancer

"VSports" E Josue Ruiz et al. J Exp Med. .

V体育官网 - Abstract

Lung squamous cell carcinoma (LSCC) and adenocarcinoma (LADC) are the most common lung cancer subtypes. Molecular targeted treatments have improved LADC patient survival but are largely ineffective in LSCC. The tumor suppressor FBW7 is commonly mutated or down-regulated in human LSCC, and oncogenic KRasG12D activation combined with Fbxw7 inactivation in mice (KF model) caused both LSCC and LADC. Lineage-tracing experiments showed that CC10+, but not basal, cells are the cells of origin of LSCC in KF mice VSports手机版. KF LSCC tumors recapitulated human LSCC resistance to cisplatin-based chemotherapy, and we identified LUBAC-mediated NF-κB signaling as a determinant of chemotherapy resistance in human and mouse. Inhibition of NF-κB activation using TAK1 or LUBAC inhibitors resensitized LSCC tumors to cisplatin, suggesting a future avenue for LSCC patient treatment. .

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Figures (VSports注册入口)

Figure 1.
Figure 1.
Biallelic inactivation of Fbxw7 and KRasG12D activation in the adult mouse lung leads to LSCC and LADC formation. (A) Representative human lung LADC (i–iv) and LSCC (v–viii) tumors and control lung sections stained with FBW7 antibodies. Bars, 20 µm. (B) Quantification of FBW7 protein staining in human LADC and LSCC tumors as in A. n = 26 LADC, 35 LSCC. (C) Biallelic inactivation of Fbxw7 and KRasG12D activation by intratracheal (IT) delivery of Ad5-CMV-Cre virus in the adult mouse lung as a model of NSCLC. (D) KF model develops LSCC (CK5+) and LADC (TTF1+) tumors. Sections representative of six animals. (E) Quantification and localization of mouse lung LADC and LSCC tumors in the KF model. n = 15 lungs. Plots indicate mean ± SD. (F) Human and mouse NSCLC samples were stained with biomarkers used clinically to distinguish LADC (TTF1) from LSCC (CK5 and ΔNp63) tumors. Bars, 100 µm (columns 1 and 4); 20 µm (columns 2 and 5). Sections representative of six animals. (G) Heat map of RNASeq data showing relative expression of LSCC genes in LADC and LSCC tumors from n = 3 mice of the KF genotype. Gene set shown is selected from gene sets up-regulated in Lkb1f/f; Ptenf/f LSCC (Xu et al., 2014) and in LSL-Sox2; Ptenf/f; Cdkn2abf/f LSCC and human LSCC (Ferone et al., 2016). Genes are ordered according to z-score. See also Fig. S1.
Figure 2.
Figure 2.
Lung LSCC originates from CC10+ luminal cells in the KF model. (A) Immunofluorescent staining for CC10, FoxJ1, Sftpc, and GFP in CK19-CreERT; R26-LSL-YFP mouse lung sections at 1 wk after induction. Images representative of three animals. Bars, 20 µm. Tam, tamoxifen. (B) Ciliated FoxJ1+ cells were targeted by intratracheal (IT) delivery of Lenti-FoxJ1-Cre virus to KF animals. The graph shows the quantification and localization of lung tumors produced. n = 12 lungs. Plots indicate mean ± SD. (C) Club CC10+ cells were targeted by intratracheal delivery of Ad5-CC10-Cre virus to KF animals. The graph shows the quantification and localization of lung tumors produced. n = 17 lungs. Plots indicate mean ± SD. (D) Scheme for analysis of cell proliferation in CK19-CreERT; KRasG12D; Fbxw7f/f; R26-LSL-YFP (CKFY) mice. (E) Lung serial sections from CKFY mice were immunostained for CC10, CK5, and EdU at 3, 7, 14, 21, and 28 d after tamoxifen injection. Arrows indicate proliferating cells. Arrowheads indicate double CK19+CK5+ cells. Images representative of five animals. Bars, 20 µm. (F) Quantification of proliferating CC10+ and CK5+ cells at the indicated time points. Graph indicates mean ± SD of five animals. See also Figs. S1, S2, and S3.
Figure 3.
Figure 3.
LSCC tumors are resistant to the chemotherapeutic agent cisplatin. (A) LADC and LSCC cells were isolated from CKFY mice 4 wk after tamoxifen injection, and CK5 and CK14 expression was analyzed by qPCR and immunostaining. n = 2 independent isolations. Bars, 20 µm. (B) Sftpc and TTF1 expression in CKFY LADC and LSCC cells (n = 2 independent isolations) was analyzed by qPCR and immunostaining. Bars, 20 µm. (C) Top: Scheme summarizing the protocol used to isolate LADC and LSCC cells from CKFY mice and to validate their tumoral properties in nude mice. Bottom: Representative IHC staining of lung tumor sections derived from isolated LADC and LSCC tumor cells (n = 2 independent isolations) after intratracheal (IT) injection. CKFY-derived, LSCC, and LADC tumor cells were identified by GFP, CK5, and TTF1, respectively. Bars, 100 µm. Tam, tamoxifen. (D) LADC, but not LSCC, cells from CKFY mice and human tumor cell lines are highly sensitive to cisplatin (CPPD) treatment (n = 4 independent experiments). (E) 9–11 wk after Ad5-CMV-Cre infection, KF mice were treated with PBS or cisplatin (7 mg/kg) and monitored for response. (F) In vivo imaging by micro-computed tomography (micro-CT) of the lungs of animals treated as in E after 4–5 wk of treatment. Images representative of two animals. H, heart; dotted line, tumor mass. (G) Lung histology of animals treated as in E, showing both LADC (TTF1+) and LSCC (ΔNp63+) tumors in mice receiving vehicle but only LSCC in mice receiving cisplatin. Images representative of two animals. (H) Quantification of LADC and LSCC tumors per animal in control and cisplatin-treated mice. n = 2 animals per condition. Plots indicate mean and range; Student’s one-tailed t test was used to calculate P values (*, P = 0.0305). See also Fig. S4.
Figure 4.
Figure 4.
LUBAC expression and linear ubiquitylation are increased in LSCC compared with LADC cells. (A) Relative mRNA expression of LUBAC components in LSCC and LADC murine cells from KF mice, measured by real-time PCR. Student’s two-tailed t test was used to calculate P values (***, P ≤ 0.0001). Graph shows mean + SD of six experiments. (B) Immunoblots showing the abundance of the three LUBAC components HOIP (RNF31), HOIL-1, and Sharpin, in LADC and LSCC cells from the KF mouse model. Blots representative of four independent experiments. MW, molecular weight. (C) TCGA data from http://www.cbioportal.org showing frequency of alterations in genes encoding LUBAC components in human LSCC and LADC. n = 230 LADC, 179 LSCC samples. (D) Relative mRNA expression of LUBAC components in LSCC (n = 19) and LADC (n = 11) patient samples from the Cordoba Biobank, measured by real-time PCR. The P value was calculated using the Student’s t test (*, P = 0.036; **, P = 0.0029). Plots indicate mean ± SEM. (E) Immunoblots of different polyubiquitin chains in whole-cell lysates from untreated (no exogenous stimulation) or TNFα-stimulated LADC and LSCC cells from KF mice. (F) TNFR1 complex I immunoprecipitated (IP) from KF LADC and LSCC cells using Flag-tagged TNFα under nondenaturing conditions and immunoblotted with Met1-Ub–specific antibodies. Blots representative of two independent experiments. WB, Western blot. See also Fig. S4.
Figure 5.
Figure 5.
Mouse and human LSCC tumors show activated NF-κB signaling. (A) Western blots showing LSCC cells from KF mice have a higher baseline level of phospho-p65 and a lower level of IκBα. Blots representative of two independent experiments. MW, molecular weight. (B) Immunofluorescent staining on LADC and LSCC primary tumor cells (top, representative of n = 3 independent isolations) and IHC staining of LADC and LSCC tumors (bottom, representative of n = 3 animals) from KF mice showing increased nuclear localization of p65 in LSCC. (C) LSCC cells have a higher baseline level of NF-κB–dependent target gene (CCL2 and TNFα) expression and respond more following TNFα stimulation when compared with LADC cells. Relative mRNA levels were measured by real-time PCR following TNFα stimulation (10 ng/ml) of LADC and LSCC cells for the indicated times. Graph represents mean (+ SD) of three independent experiments performed in duplicate. (D) Western blots showing that human LSCC tumor cell lines exhibit higher levels of p65 activation compared with LADC cell lines. Ratio of phospho-p65 to total p65 normalized to GAPDH is given below. Samples were run in parallel, and identical exposures were used. (E) Quantification of results in D. Student’s one-tailed t test was used to calculate P values (**, P = 0.0095). Plots indicate mean ± SEM. (F) Human LSCC tumors exhibit higher levels of p65 activation compared with LADC samples. Ratio of phospho-p65 to total p65 normalized to actin is given below. Samples were run in parallel, and identical exposures were used. (G) Quantification of results in F and from a total of 25 LADC and 31 LSCC patient tumors. Student’s two-tailed t test was used to calculate P values (***, P = 0.0002). Plots indicate mean ± SEM. (H) Relative mRNA expression of CCL2 and TNFα in LSCC (n = 19) and LADC (n = 11) patient samples from the Cordoba Biobank, measured by real-time PCR. Student’s two-tailed t test was used to calculate P values (*, P = 0.01; **, P = 0.0049). Plots indicate mean ± SEM. See also Fig. S4.
Figure 6.
Figure 6.
Inhibition of LUBAC or TAK1 sensitizes LSCC cells to cisplatin. (A) Both 5Z-7 and gliotoxin inhibit NF-κB signaling, as measured by phospho-p65 (Ser536). Blots represent two independent experiments. MW, molecular weight. (B) Expression of NF-κB–dependent target genes (CCL2 and TNFα) in murine LSCC cells is decreased in the presence of the TAK1 inhibitor 5Z-7 or the LUBAC inhibitor gliotoxin. Relative mRNA levels measured by real-time PCR. Data are mean + SD of three independent experiments performed in triplicate. (C) LSCC cells are highly sensitive to gliotoxin treatment, and gliotoxin sensitizes cells to cisplatin. Data are mean + SD of two independent experiments performed in duplicate. ***, P < 0.0001 versus vehicle, two-way ANOVA. (D) siRNA-mediated knockdown of the LUBAC components HOIP and HOIL-1 decreases LSCC cell resistance to cisplatin. Graph shows mean + SD of three independent experiments performed in triplicate. ***, P < 0.0001 versus vehicle, one-way ANOVA. (E) Validation of HOIL-1 knockdown in LSCC cells using two independent siRNAs. Graphs represent mean + SEM of three independent experiments. (F) Validation of HOIP knockdown in LSCC cells using three independent siRNAs. Graphs represent mean + SEM of three independent experiments. (G) In vitro LSCC and LADC cells are highly sensitive to cisplatin (CPPD) and 5Z-7-oxozeaenol combination treatment. Data are mean + SD of two independent experiments performed in duplicate. ***, P < 0.0001 versus vehicle, two-way ANOVA. (H) Validation of TAK1 knockdown in LSCC cells using three independent siRNAs. Graphs represent mean + SEM of three independent experiments. (I) siTAK1 LSCC cells are sensitive to cisplatin (CPPD), and addition of 5Z-7 does not affect CPPD sensitivity in the presence of TAK1 knockdown. P values calculated using one-way ANOVA test. Graph shows mean + SD of two independent experiments performed in quadruplicate. ***, P < 0.0001 versus vehicle, one-way ANOVA. See also Fig. S5.
Figure 7.
Figure 7.
Combination treatment with cisplatin and TAK1 inhibitor or gliotoxin sensitizes LSCC tumors. (A) In vivo tumor graft growth curves of LADC and LSCC cells subcutaneously injected in both flanks of athymic NU/NU mice. Mice with palpable tumors were treated with i.p. injections as follows. Top: Cisplatin (CPPD; n = 3), 5Z-7-oxozeaenol (n = 3), 5Z-7-oxozeaenol+cisplatin (n = 3), or vehicle (n = 3). Bottom: Cisplatin (n = 3), gliotoxin+cisplatin (n = 4), or vehicle (n = 4). Data are mean ± SEM of the tumor volumes. P values calculated from two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 versus vehicle. (B) Scheme depicting experimental design for in vivo test of cisplatin (3.5 mg/kg) alone or in combination with 5Z-7-oxozeaenol (7 mg/kg) or gliotoxin (2.5 mg/kg). (C) Quantification of LADC and LSCC tumors per animal in control, cisplatin-treated, and 5Z-7 combination-treated KF mice. P values calculated using two-way ANOVA (n = 6 vehicle and cisplatin, n = 8 combination). **, P = 0.009; ***, P < 0.001. Plots show mean ± SEM. (D) Histological analysis of LADC and LSCC tumors in control, cisplatin-treated, and combination-treated animals. Representative of animals in C (vehicle, cisplatin, and 5Z-7 combination) and E (gliotoxin combination). Bars, 50 µm. (E) Quantification of LADC and LSCC tumors per animal in control, cisplatin-treated, and gliotoxin combination–treated KF mice. P values calculated using two-way ANOVA (n = 3 each treatment; *, P = 0.031; **, P = 0.0013). Plots show mean ± SEM. See also Fig. S5.
Figure 8.
Figure 8.
Combination treatment with cisplatin and TAK1 inhibitor increases survival even with advanced tumors. (A) Scheme depicting experimental design for end-stage survival analysis of control, cisplatin-treated, and combination-treated KF mice. IT, intratracheal. (B) Kaplan-Meier curve showing effect of cisplatin (CPPD; 3.5 mg/kg) or cisplatin plus 5Z-7-oxozeaenol (7 mg/kg) treatment on survival of tumor-bearing KF mice (n = 4 vehicle and cisplatin, n = 6 combination). P value calculated using log-rank (Mantel–Cox) test (**, P = 0.0011). See also Fig. S5.
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