Twist1-Induced Epithelial Dissemination Requires Prkd1 Signaling

Abstract

Dissemination is an essential early step in metastasis but its molecular basis remains incompletely understood. To define the essential targetable effectors of this process, we developed a 3D mammary epithelial culture model, in which dissemination is induced by overexpression of the transcription factor Twist1. Transcriptomic analysis and ChIP-PCR together demonstrated that protein kinase D1 (Prkd1) is a direct transcriptional target of Twist1 and is not expressed in the normal mammary epithelium. Pharmacologic and genetic inhibition of Prkd1 in the Twist1-induced dissemination model demonstrated that Prkd1 was required for cells to initiate extracellular matrix (ECM)-directed protrusions, release from the epithelium, and migrate through the ECM. Antibody-based protein profiling revealed that Prkd1 induced broad phosphorylation changes, including an inactivating phosphorylation of β-catenin and two microtubule depolymerizing phosphorylations of Tau, potentially explaining the release of cell-cell contacts and persistent activation of Prkd1. In patients with breast cancer, TWIST1 and PRKD1 expression correlated with metastatic recurrence, particularly in basal breast cancer. Prkd1 knockdown was sufficient to block dissemination of both murine and human mammary tumor organoids. Finally, Prkd1 knockdown in vivo blocked primary tumor invasion and distant metastasis in a mouse model of basal breast cancer. Collectively, these data identify Prkd1 as a novel and targetable signaling node downstream of Twist1 that is required for epithelial invasion and dissemination. SIGNIFICANCE: Twist1 is a known regulator of metastatic cell behaviors but not directly targetable. This study provides a molecular explanation for how Twist1-induced dissemination works and demonstrates that it can be targeted. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/2/204/F1.large.jpg.

Figure 1.. Identification of targetable molecular requirements for Twist1-induced epithelial dissemination.

(a) Schematic representation of our experimental pipeline. Mammary organoids from Twist1-inducible transgenic mice were cultured without (Twist1-Off) or with (Twist1-On) doxycycline to induce Tet-activated Twist1 expression. Differential RNA-sequencing (RNAseq) of these organoids identified Twist1-upregulated (red) and downregulated (blue) genes represented here as a volcano plot, where the reported p-value is corrected for multiple testing (dataset, p-values and fold changes obtained from (5)). The Twist1-upregulated gene set was prioritized for functional investigation (full list of small molecules and corresponding targets can be found in Supplementary Table 1). Each small molecule was tested at 5 concentrations (0, 10, 100, 1000, 10000 nM) in Twist1-On organoids. For each dose, the number of disseminated cells per organoid was normalized to that of the vehicle control (0 nM), then curve-fitted with a Hill dose-response function. (b) Plot representing IC50 and dissemination at 1 μM for individual small molecules (and corresponding target proteins) used in the Twist1-induced dissemination assay (a). For each compound, n ≥ 20 organoids per mouse per dose, r = 3 mice, Tested molecules include Twist1-predicted inhibitors (red) and non-Twist1-predicted inhibitors (black, detailed in Supplementary Table 1). *, FDA-approved drugs. ^#, data values from (6). Error bars represent SEM. To the left of the y-axis, representative DIC micrographs display the range of dissemination inhibition. Red arrowheads indicate disseminated cells. Scale bars, 50 μm. (b’) Potent inhibitors with IC50 < 1000 nM and dissemination at 1 μM < 33.3% (yellow box).

Figure 2.. Prkd1 is selectively required in Twist1-induced dissemination.

(a) Volcano plot showing differential expression of Prkc and Prkd gene family members in Twist1-On relative to Twist1-Off organoids (from dataset shown in Fig. 1a and previously published in (5)). (b) Western blots showing Prkd1, Prkd2, Prkca, Pkrcb1, Twist1, and β-tubulin protein expression in Twist1-Off and Twist1-On organoids. (b’,b”) Bar graph (mean ± SEM) showing protein expression fold change for Prkd1 (n = 5 mice) and Twist1 (n = 6 mice), respectively, in Twist1-Off and Twist1-On organoids. Statistical test: Mann-Whitney. ** p < 0.01. (c,c’) Confocal micrographs of Prkd1 immunofluorescence staining in Twist1-Off and Twist1-On organoids, respectively. Scale bar, 20 μm. (c”) Dot plot (with median) showing Prkd1 expression levels in Twist1-Off organoids, and Twist1-On organoids and disseminated cells. For Twist1-Off, r = 2 mice, n = 3 organoids. For Twist1-On, r = 2 mice, n = 11 organoids (52 disseminated cells). Statistical test: Kruskal-Wallis. * p < 0.01; **** p < 0.0001. (d) Bar graph (mean ± SEM) showing enrichment of Twist1 ChIP region in the Prkd1 gene quantified using qPCR. Data is represented as fold-change over input (non-IP) control. (e,f) DIC micrographs of Twist1-Off organoids cultured for 7 days and treated with different doses of Gö-6976 or Sorafenib. Scale bars, 50 μm. (e’,f’) Paired dot plots showing fold change in projected surface area of organoids in (e) and (f), respectively. For each drug, r = 3 mice, n = 25–105 organoids per dose. Statistical test: Kruskal-Wallis. ns, p > 0.05; **** p < 0.0001. (g) DIC micrographs of Twist1-On organoids lentivirally transduced with non-target (NT, control) shRNA and Prkd1 shRNA clones #1 or #2. Red arrowheads indicate disseminated cells. Scale bar, 50 μm. (h) Western blot and bar graph (mean ± SEM) showing Prkd1 protein expression in control (NT) or Prkd1 knockdown (sh#1, sh#2) Twist1-On organoids represented in (g). Data is collected from r = 2 experiments. (i) Whisker plot (Tukey method) showing dissemination in control (NT) or Prkd1 knockdown (sh#1, sh#2) Twist1-On organoids represented in (g). Data is collected from r = 3 mice; n = 65 organoids (NT), 39 organoids (sh#1), 22 organoids (sh#2). Statistical test: Kruskal-Wallis. *, p < 0.05; ****, p < 0.0001. (j) Whisker plot (10–90th percentile) showing projected organoid surface area in control (NT) or knockdown (sh#1, sh#2) Twist1-On organoids represented in (g). Data collected from r = 3 mice; n = 20 organoids (NT), 14 organoids (sh#1), 17 organoids (sh#2) Statistical test: Kruskal-Wallis. ns, p > 0.05.

Figure 3.. Prkd1 expression and activity are required for epithelial invasion, loss of cell-cell adhesion, and persistent migration.

(a,b) DIC micrographs from timelapse imaging of Twist1-On organoids treated with vehicle (a) or Gö-6976 (b). Zoomed insets are presented for (a) in (a’,a”) and for (b) in (b-b”). Magenta and cyan arrowheads indicate ECM-invading cells and early disseminated cells, respectively. Scale bars, 50 μm. (c) Stacked bar graph (mean with 95% confidence interval) showing quantification of the percentage of organoids with (magenta) or without (white) ECM-invading cells from (a,b). (d) Whisker plot (min to max) showing quantification of the number of disseminated cells per organoid from timelapse movies represented in (a,b). Data for (c,d) was collected from r = 3 mice, n = 47 (Veh), 33 (Gö-6976 330 nM), 31 (Gö-6976 3.3 μM) organoids. (e,f) DIC micrographs and zoomed insets from timelapse imaging of Twist-induced organoids during a 70h interval without treatment (e’-e”, f’-f”) then a 70h interval with treatment with vehicle (e”-e”’) or Gö-6976 (f”-f’’’). Complete cell migration tracks for the 70h untreated phase are represented in green. Full cell migration tracks for the 70h vehicle treated or Gö-6976-treated phase are represented in yellow and red, respectively. Scale bars, 50 μm. (g) Paired dot plot showing mean cell velocities of individual disseminating cells before or after treatment with vehicle or Gö-6976, for data represented in (e,f). Data was collected from r = 3 mice, n = 60 cells (Veh), 30 cells (330 nM), 18 cells (3.3 μM). Statistical test: Kruskal-Wallis. ns, p > 0.05; ***, p < 0.001; ****, p < 0.0001. (h) Whisker plot (min-max) showing quantification of the number of disseminated cells per organoid in the 70h interval following treatment with vehicle or Gö-6976 (f’-f”). Statistical test: Kruskal-Wallis. **** p < 0.0001.

Figure 4.. Identification of Twist1-upregulated and Prkd1-maintained phosphorylations in disseminating organoids.

(a) Experimental outline showing Twist1-inducible organoids utilized for phosphoantibody microarray. Four culture conditions are tested: Twist1-Off vehicle treated organoids (undergo branching morphogenesis), Twist1-On organoids (undergo dissemination), and Twist1-On organoids treated with either kb-NB142–70 (1 μM) or Gö-6976 (1 μM) (dissemination inhibited). (b) Linear plot showing 81 protein phosphorylations (out of 359 tested) identified by phosphoantibody microarray to be increased in Twist1-On compared to Twist1-Off organoids (red arrow), and decreased in Prkd1-inhibited compared to vehicle-treated Twist1-On organoids (blue arrows). (b’) Heatmap of the protein phosphorylations identified in (b). Color scale bar indicates fold change.

Figure 5.. Twist1 induces Prkd1-dependent phosphorylation of Tau and β-catenin.

(a) DIC micrographs of Twist1-On organoids treated with vehicle, Gö-6976, kb-NB142–70, and Gö-6983, taxol, and nocodazole alone or in combinations (as specified). Red arrowheads indicate disseminated cells. Scale bars, 100 μm. (b) Whisker plot (Tukey method) showing dissemination quantification of Twist1-On organoids represented in (a). Data is collected from r = 3 mice, n = 581 (Veh) and 38–155 (drug treatment) organoids. Statistical test: Kruskal-Wallis. ns, p > 0.05; **, p < 0.001; ****, p < 0.0001. (c) Confocal micrographs of fluorescently labeled total β-catenin (magenta), non-phosphorylated S33/S37/T41 (non-pS33/S37/T41) β-catenin (white), and F-actin (red) in organoids treated as indicated in (a). Dotted lines outline organoid-matrix border (yellow) or disseminated cell contour (green). Scale bars, 5 μm. (c’,c’’) Whisker plots (min-max) showing relative fluorescence intensity quantification of total β-catenin (c’) and non-pS33/S37/T41) β-catenin (c’’) from micrographs represented in (c). Data is collected from n = 3 mice, r = 6–8 organoids per condition. Statistical test: Kruskal-Wallis. ns, p > 0.05; **, p < 0.001.

Figure 6.. Prkd1 is required for mammary tumor organoid invasion and correlates with metastatic outcome in patients.

(a) Experimental outline for mammary organoid culture from the Twist1-inducible model and the two breast cancer models MMTV-PyMT and C3(1)-TAg. (a’) Western blots showing Prkd1 protein expression (and β-tubulin as a control) in organoids from mouse models shown in (a). (b) DIC micrographs showing C3(1)-TAg tumor organoids treated with vehicle or Gö-6976 (1 μM). Blue brackets indicate invasion strands. Red arrowheads indicate disseminated cells. Scale bas, 100 μm. (b’-b’’) Bar graph (mean ± SEM) showing quantification of the number of invasive strands (b’) and disseminated cells (b’’) per organoid. Data is collected from r = 3 mice, n = 39–57 organoids per dose. Statistical test: Kruskal-Wallis. ns, p > 0.05; ** p < 0.01; **** p < 0.0001. (c) Heatmap showing mRNA co-expression of TWIST1 with either PRKD1, PRKCA, or PRKCB in human breast tumors (data from bc-GenExMiner). The color scale indicates Pearson’s r, and the exact value is indicated in each box. (d) Experimental outline for organotypic culture of surgically-isolated human breast tumors. (e) Representative DIC micrographs of human tumor organoids treated with Gö-6976, kb-NB142–70, or Gö-6983. Organoid outline is depicted by the yellow line. Disseminated cells are indicated by red arrowheads. Scale bar, 100 μm. (e’) Bar graph (median ± interquartile range) showing quantified dissemination of human tumor organoids treated with increasing doses of drugs used in (e). Statistical test: Kruskal-Wallis. ns, p > 0.05; **** p < 0.0001. (f,g,h) Kaplan–Meier plots showing distant-metastasis free survival (DMFS) in a general population of 1746 breast cancer patients and (f’,g’,h’) or in a subset of 232 patients with basal breast cancer. Patient groups were separated based on TWIST1 and/or PRKD1 mRNA expression. HR, hazard ratio.

Figure 7.. Prkd1 is required for C3(1)-TAg tumor invasion and metastasis in vivo.

(a) Experimental outline for lentiviral shRNA transduction in C3(1)-TAg;mTmG tumor organoids followed by their transplantation into recipient NSG mice with pre-cleared mammary fat pads. (b) Western blot validating Prkd1 knockdown in C3(1)-TAg;mTmG tumor organoids prior to orthotopic transplantation. (c,d) Micrographs of transplanted C3(1)-TAg;mTmG tumor cryosections from (a) with DAPI-stained nuclei (blue). Tumor cell membranes expressing mT (membrane Tomato) are shown in red. Scale bar, 100 μm. (c’-d’) Zoomed insets of micrographs in (c-d) showing tumor-stroma border (yellow line). (e,f) Macrographs of whole lungs of tumor-receiving mice from (a) GFP displaying metastatic colonies (GFP-expressing puncta). Scale bar, 500 μm. (e’,f’) Zoomed insets from (e,f) with metastatic colonies indicated by yellow arrowheads. (g) Dot plot (with median bar) showing the quantification of lung metastases as represented in (e,f).