Intra-tumoral YAP and TAZ heterogeneity drives collective NSCLC invasion that is targeted by SUMOylation inhibitor TAK-981

Abstract

Non-small cell lung cancer (NSCLC) collective invasion is supported by cooperativity of proliferative (follower) and invasive (leader) cells. H1299-isolated follower cells exhibit higher Yes-associated protein (YAP) expression, while leader cells were found to express elevated transcriptional coactivator with PDZ-binding motif (TAZ/WWTR1) expression. Suppressing TAZ (not YAP) in leader cells reduced invasion. TAZ-regulated leader cell invasion is associated with activation of the EGFR-PI3K-AKT axis. NSCLC patient samples also demonstrated heterogeneity in YAP and TAZ expression. YAP and TAZ regulate proliferation of follower and leader cells. Our results highlight the need to inhibit both YAP and TAZ to effectively target their regulation of collective invasion. We identify that the SUMOylation inhibitor TAK-981 reduces YAP and TAZ expression, decreasing tumor burden and metastasis in a murine NSCLC model. Our study reveals an intra-tumoral division of labor, driven by differential YAP and TAZ expression, which can be effectively targeted with TAK-981 for NSCLC therapy.

Keywords: Cancer; Cell biology; Microenvironment.

Graphical abstract

Figure 1

YAP and TAZ are differentially expressed in H1299 leader (L) and follower (F) cells, and TAZ regulates leader cell migration and collective invasion with follower cells (A) YAP and TAZ expression evaluated and quantified in nuclear and cytosolic fractions of leader and follower cells using Lamin B1 and GAPDH as loading controls. Data are mean ± SEM from three independent experiments. (B) Quantitation of total YAP and TAZ protein levels, normalized to loading control. Data are mean ± SEM from six independent experiments. (C) Representative immunofluorescence images of endogenous YAP (red) and TAZ (green) merged with nuclear stain DAPI (blue) in leaders and follower cells (scale bar, 10 μm). (D) YAP and TAZ expression in non-targeting (control) or YAP/TAZ shRNA-transduced leader and follower cells assessed by western blot analysis. (E) Cells from (D) evaluated for migratory potential in a scratch assay at indicated time points with gap closure quantified relative to respective controls (n = 3; scale bar, 100 μm). (F–I) Proliferation and invasive potential of spheroids generated from control or YAP or TAZ-KD (F and G) leader cells, (H and I) follower cells, and (J and K) mixes of 10% leader cells with 90% follower cells (L:F, 1:9) assessed for 3D invasive area (n = 4; scale bar, 100 μm). (L) Expression levels of indicated proteins evaluated in lysates from (B). Representative blots from one of three independent experiments are presented using β-actin as loading control. Data are represented as mean ± SEM. An ordinary one-way ANOVA with a Tukey’s multiple comparisons test was used to determine significance compared to control or as indicated. ∗p < 0.05, ∗∗p < 0.01 ∗∗∗p < 0.001 compared to the indicated group in post hoc tests.

Figure 2

YAP and TAZ exhibit intra-tumoral heterogeneity in NSCLC metastases, and TAZ expression correlates with fibronectin expression in NSCLC (A) Representative IHC images of YAP and TAZ, from 3 metastatic human non-small cell lung cancer samples. Arrows show neoplastic cells with more intense staining. Arrowheads show neoplastic cells with minimal to no staining. Scale bar, 20 μm. (B) Correlation analysis of the TCGA-Broad institute (Firehose LUAD dataset) cohort (n = 365) for fibronectin protein with YAP or TAZ expression. A Spearman correlation of 0.2852 (∗∗∗∗) and 0.03 (not significant) was determined for TAZ and YAP association with FN, respectively.

Figure 3

TAZ and EGFR are reciprocally regulated in leader cells (A) Correlation analysis of EGFR mRNA and YAP and TAZ mRNA expression in our institutional Winship molecular profiling (2020) dataset. A Spearman correlation of 0.4260 (∗∗∗∗) and 0.5205 (∗∗∗∗) for TAZ and YAP, respectively, vs. EGFR expression was noted (n = 343). (B) Whole cell lysate of leader and follower non-targeting (control) or YAP/TAZ-KD cells from Figure 1B evaluated for EGFR expression. Data are mean ± SEM from three independent experiments. (C) Whole cell lysates of leader cells, leader TAZ-KD cells, or L TAZ-KD cells overexpressing (OE) YAP or TAZ were evaluated for indicated proteins. Representative blots from one of three independent experiments presented. (D) EGFR mRNA expression in YAP or TAZ OE leader and follower cells. (E) Examination of the effect of erlotinib (72 h treatment at indicated doses) on indicated proteins isolated from whole cell lysates of leader and follower cells. Quantitation of the immunoblot analyses (F) for YAP and TAZ, normalized to the loading control. Densitometric quantification from n = 3 blots represented as mean ± SEM. (F and G) Effect of erlotinib on invasive potential and proliferation of spheroids generated with leader cells, follower cells, or 90% follower and 10% leader cells mixes (L: F, 1:9.) with the quantification of invasive area. (n = 4; scale bar, 100 μm). Data are represented as mean ± SEM. An ordinary one-way or two-way ANOVA with a Tukey’s multiple comparisons test was used to determine significance compared to control or as indicated. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the indicated group.

Figure 4

TAZ-regulated collective invasion is rescued by AKT OE (A and B) Whole cell lysate of leader and follower non-targeting (control) or YAP/TAZ shRNA KD cells (cells from Figure 1D) or leader TAZ-KD cells OE YAP or TAZ evaluated for expression of indicated proteins using β-actin as a loading control. Representative blots from one of three independent experiments presented. pAKT/AKT quantification data represented mean ± SEM from three independent experiments. (C–F) Invasion and proliferation evaluated (C and D) in spheroids generated from leader cells or TAZ-KD leader cells OE AKT1 or AKT2, or (E and F) in mixes with follower cells (1:9 ratio) with corresponding quantification of invasive area. (n = 4; scale bar, 100 μm). Data are represented as mean ± SEM. An ordinary one-way ANOVA with a Tukey’s multiple comparisons test was used to determine significance compared to control or as indicated. ∗p < 0.05 ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the indicated group.

Figure 5

AKT-GS3β-βTrCP axis contributes to differential TAZ expression in follower and leader cells (A) Evaluation of YAP and TAZ mRNA expression in leader and follower cells. (B) YAP, TAZ, and β-actin expression in leader and follower cells treated with indicated doses of MG132 for 16 h. (C) Whole cell lysates of leader and follower cells analyzed for βTrCP, AKT, GSK3β, and their phosphorylation status using β-actin as a loading control. βTrCP expression levels were quantified, and data represent mean ± SEM from n = 3. (D) Indicated proteins evaluated and quantified in leader and follower cells treated with indicated doses of AKTi for 24 h. Data represent mean ± SEM from n = 3. (E) Whole cell lysates of leader and follower cells expressing non-targeting control or βTrCP-directed shRNA evaluated for the indicated proteins. (F) Evaluation of indicated proteins in leader and follower cells overexpressing βTrCP. Representative blot from one of three independent experiments presented. (G) Schematic showing the mechanistic basis for differential expression of TAZ in leader and follower cells, created with Biorender.com. Data are represented as mean ± SEM. An ordinary one-way ANOVA with a Tukey’s multiple comparisons test or t test was used to determine significance compared to control or as indicated. ∗p < 0.05 ∗∗p < 0.01 compared to the indicated group in the post hoc tests.

Figure 6

SUMOylation inhibitor TAK-981 suppresses YAP and TAZ SUMOylation and YAP/TAZ expression (A) Leader and follower cells treated with indicated doses of TAK-981 for 72 h were evaluated for YAP and TAZ. Expression levels normalized to β-actin (loading control) are presented. Data are presented as means ± SEM from three independent repeats. One representative experiment of an n = 3 is shown. (B) Immunoprecipitates of YAP and TAZ from whole cell lysates leader and follower cells treated +/− TAK-981 for 6 h were evaluated for SUMO-1. (C and D) Leader and follower cells treated with TAK-981 for 24 h followed by analysis of whole cell lysates for indicated proteins. Quantification of indicated protein represented as mean ± SEM from three independent repeats. (E and F) Spheroids generated with leader cells, follower cells, or mixes of 90% follower cells and 10% leader cells (L:F, 1:9) treated with either DMSO or indicated doses of TAK-981 were evaluated for invasive area with quantification of invasive area (n = 4; scale bar, 100 μm). (G) Schematic depiction of YAP- and TAZ-driven regulation of intra-tumoral heterogeneity supporting collective invasion that is inhibited by TAK-981, created with Biorender.com. Data are represented as mean ± SEM. An ordinary one-way ANOVA with a Tukey’s multiple comparisons test or t test was used to determine significance compared to control or as indicated. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the indicated group in the post hoc tests.

Figure 7

TAK-981 inhibits the in vitro proliferation and collective invasion of NSCLC lines and in vivo tumor growth and metastases in a preclinical model of NSCLC (A) Indicated proteins and β-actin (loading control) were assessed in A549, H460, PC9, H596, H23, and H1975 cells after 72 h of TAK-981 treatment. (B) Spheroids generated with indicated cell lines treated with either DMSO or TAK-981 (500 nM) were evaluated for invasive area and proliferation with quantification of invasive area (n = 5; scale bar, 100 μm). (C) Experimental treatment strategy used in H1299-NSG murine tumor model. Tumors were subcutaneously injected in NSG mice (n = 10 mice per group) and randomized to two groups when tumors were palpable (approximately three weeks). Mice were treated with vehicle or 25 mg/kg TAK-981 thrice weekly. (D) Bright-field images of tumors isolated from vehicle- and TAK-981-treated mice. (E and F) (E) Mean tumor weight and (F) mean tumor volume of mice treated with vehicle or TAK-981. (G) Western blot to assess expression level of indicated proteins in tumors from vehicle- and TAK-981-treated mice. (H) Representative images of lung and liver macro mets (indicated by arrows) identified from vehicle-treated groups. TAK-981-treated lung indicates (with arrow) a micro met while a liver section shows EMH and fibrin overlaying the capsule. (I) Percentage lung metastases was calculated by quantification of total macro and micro mets area normalized to total area in H&E-stained lung sections from vehicle- and TAK-981-treated mice (n = 10). Data are represented as mean ± SEM. t test was used to determine significance compared to control or as indicated. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 compared to the indicated group in the post hoc tests.