Cell phenotypic plasticity requires autophagic flux driven by YAP/TAZ mechanotransduction

Abstract

Autophagy, besides ensuring energy metabolism and organelle renewal, is crucial for the biology of adult normal and cancer stem cells. However, it remains incompletely understood how autophagy connects to stemness factors and the nature of the microenvironmental signals that pattern autophagy in different cell types. Here we advance in these directions by reporting that YAP/TAZ transcriptionally control autophagy, being critical for autophagosomal degradation into autolysosomes. YAP/TAZ are downstream effectors of cellular mechanotransduction and indeed we found that cell mechanics, dictated by the physical property of the ECM and cytoskeletal tension, profoundly impact on autophagic flux in a YAP/TAZ-mediated manner. Functionally, by using pancreatic and mammary organoid cultures, we found that YAP/TAZ-regulated autophagy is essential in normal cells for YAP/TAZ-mediated dedifferentiation and acquisition of self-renewing properties. In tumor cells, the YAP/TAZ-autophagy connection is key to sustain transformed traits and for acquisition of a cancer stem cell state by otherwise more benign cells. Mechanistically, YAP/TAZ promote autophagic flux by directly promoting the expression of Armus, a RAB7-GAP required for autophagosome turnover and whose add-back rescues autophagy in YAP/TAZ-depleted cells. These findings expand the influence of YAP/TAZ mechanotransduction to the control of autophagy and, vice versa, the role of autophagy in YAP/TAZ biology, and suggest a mechanism to coordinate transcriptional rewiring with cytoplasmic restructuring during cell reprogramming.

Keywords: YAP/TAZ; autophagy; cell plasticity; mechanotransduction.

Fig. 1.

YAP/TAZ control autophagic flux by regulating autophagosome degradation. (A and B) Immunoblot analysis for YAP/TAZ and LC3 in MDA-MB-231 (A) and MCF10A (B) transfected with control siRNA (siCo.) or 2 independent YAP/TAZ siRNA mixes (siY/T #1 or siY/T #2) for 48 h. The cleaved LC3 peptide and its phosphatidylethanolamine conjugated form are indicated as LC3-I and LC3-II, respectively. GAPDH serves as loading control. (C and D) R26^CAG-CreER; Yap^fl/fl; Taz^fl/fl mice carrying an ubiquitous inducible Cre recombinase were treated with tamoxifen to obtain the knockout of both YAP and TAZ floxed alleles in vivo (Yap^−/−; Taz^−/−; i.e., Y/T KO). Tamoxifen-treated Yap^fl/fl; Taz^fl/fl littermate mice (without CRE) were used as control. See also SI Appendix, Methods. Immunoblot analysis of lysates from skin (C) and pancreatic biopsies (D) shows increased levels of LC3-II in YAP/TAZ knockout mice, compared to their controls (Top). Knockout of YAP and TAZ was confirmed by Western blot (Middle). Residual YAP and TAZ proteins remain in these blots due to cells escaping recombination. GAPDH serves as loading control (Bottom). (E and F) MDA-MB-231 cells stably expressing GFP-LC3 construct (MDA-GFP-LC3) were transfected with control siRNA (siCo) or 2 independent YAP/TAZ siRNA mixes (siY/T #1, siY/T #2) for 48 h. Cells were treated with medium (−CQ) or CQ 50 μM (+CQ) for the last 4 h. E shows representative confocal images. The Insets (2×) show higher magnification of the GFP-LC3 puncta. DAPI (blue) is a nuclear counterstain. (Scale bar, 20 μm.) (F) Quantification of GFP-LC3 puncta accumulation, measured as area of GFP-LC3 puncta per cell. Bars represent mean + SEM from 4 independent experiments. (*P ≤ 0.0001, **P < 0.05, compared to −CQ siCo; 2-way ANOVA). See SI Appendix, Methods for detailed GFP-LC3 quantification method. (G and H) MDA-GFP-LC3 cells transfected as in E, were cultured in full nutrient medium (full medium) or subjected to nutrient starvation in Hank's balanced salt solution (HBSS; starved) for the last 4 h. (G) Panels are representative confocal images. (H) Quantification of GFP-LC3 puncta accumulation as in F. Bars represent mean + SEM from 3 independent experiments (§P < 0.05, §§P < 0.01, compared to full medium siCo; *P < 0.0001, **P < 0.001 compared to starved siCo; 2-way ANOVA). (I and J) MII cells stably expressing GFP-LC3 construct (MII-GFP-LC3) were infected with an empty lentiviral vector (empty) or with the indicated siRNA-insensitive doxycycline-inducible lentiviral Flag-tagged YAP constructs. Cells were transfected with either control (siCo) or YAP/TAZ siRNAs (siYAP/TAZ), treated with doxycycline and analyzed 48 h after siRNA transfection. Cells were concomitantly treated with or without CQ for the last 4 h. (I) Panels are representative confocal images. (Scale bar, 20 μm.) (J) Quantification of GFP-LC3 puncta as in F. Bars represent mean + SEM (*P < 0.01 compared to −CQ empty-infected siCo, §P < 0.01 compared to −CQ empty-infected siYAP/TAZ; 1-way ANOVA). See also SI Appendix, Fig. S1G for validation of YAP/TAZ transcriptional activity in I and J.

Fig. 2.

YAP/TAZ regulate the fusion of autophagosomes with lysosomes. (A) Schematic of autophagosome degradation during autophagic flux. Mature autophagosomes (stained by GFP-LC3; green) containing cytoplasmic materials fuse with lysosomes (stained by LAMP-1; red) to become autolysosomes. (B and C) MDA-GFP-LC3 transfected with control siRNA (siCo) or 2 independent YAP/TAZ siRNAs mixes (siY/T #1 and siY/T #2) were stained for the lysosomal membrane protein LAMP-1. Panels are representative confocal images of the colocalization between GFP-LC3 puncta (green) and LAMP1-positive vesicles (red) (B), and the corresponding quantification, scored as the ratio of the double-positive vesicles (yellow) to the total of GFP-LC3 puncta (green) per cell (C). DAPI (blue) is a nuclear counterstain. The Inset magnification is 2.5×. (Scale bar, 20 μm.) (D and E) MDA-GFP-LC3 transfected as previously described (B and C) were starved in HBSS for 4 h and stained for LAMP-1 to evaluate the colocalization between GFP-LC3 puncta (green) and LAMP-1 positive vesicles (red). Panel shows representative confocal images of GFP-LC3 (green) and LAMP-1 staining (red) (D) and the corresponding quantification of the colocalization between GFP-LC3 puncta (green) and LAMP-1 positive vesicles (red) (E). (F) Schematics of differently labeled autophagosomes (mCherry+/GFP+) and autolysosomes (mCherry+/GFP−) in MDA-mCherry-GFP-LC3. (G and H) Cells were transfected with control siRNA (siCo) or 2 independent YAP/TAZ siRNAs mixes (siY/T #1 and siY/T #2). (G) Representative confocal images of MDA-mCherry-GFP-LC3 cells treated as above. DAPI (blue) is a nuclear counterstain. The Inset magnification is 2×. (Scale bar, 20 μm.) (H) Quantification of the percentage of autophagosomes and autolysosomes scored, respectively, as the ratio of GFP+/mCherry+ puncta (autophagosomes) and GFP-/mCherry+ puncta (autolysosomes) to the total number of mCherry positive puncta (autophagosomes and autolysosomes). (C, E, and H) At least 30 cells from 3 independent experiments were scored for each condition. Data are presented as box and whisker plots: the box extends from the 25th to the 75th percentile; the line within the box represents the median; whiskers extend to show the highest and lowest values (*P < 0.0001 compared to siCo; 1-way ANOVA). (G and H) Number of autophagosomes ± SEM: siCo = 10.44 ± 2.31, siYAP/TAZ #1 = 21.92 ± 3.10, siYAP/TAZ #2 = 22.06 ± 3.14; number of autolysosomes ± SEM: siCo = 15.15 ± 2.96, siYAP/TAZ #1 = 6.65 ± 0.93, siYAP/TAZ #2 = 6.12 ± 0.95.

Fig. 3.

YAP/TAZ mechanotransduction regulates autophagy. (A) Representative confocal images of GFP-LC3 (green) and YAP/TAZ (red) in MII-GFP-LC3 cells plated on stiff (40 kPa) vs. soft (2.0 kPa) fibronectin-coated acrylamide hydrogels for 24 h. Cells were treated with vehicle (−CQ) or with CQ (+CQ) for the last 4 h. DAPI (blue) is a nuclear counterstain. (Scale bar, 20 μm.) (B) Percentage of MII-GFP-LC3 cells accumulating GFP-LC3 puncta as in A. Bars represent mean + SD from 6 different experiments (*P < 0.0001 compared to −CQ stiff; 2-way ANOVA). Cells with more than 10 GFP-LC3 puncta were scored as positive. (C) MII-GFP-LC3 were embedded as a single cell in a 3D matrix formed of Matrigel supplemented with different doses of collagen-I. Soft matrix contained 0.25 mg/mL collagen-I (soft 3D), whereas stiff matrix contained 1 mg/mL collagen-I (stiff 3D). After 5 d, cells were fixed, stained with phalloidin (red) to visualize the morphology of multicellular structures, and analyzed for the presence of GFP-LC3 puncta (green). (Scale bar, 50 μm.) (D) Quantification of GFP-LC3 puncta induced of cells experiencing a soft 3D ECM and plated as in C. At least 30 multicellular structures (>1,500 cells) from 3 independent experiments were scored for each condition; bars represent mean + SD (*P < 0.001, compared to stiff 3D; 2-tailed Student’s t test). (E and F) MII-GFP-LC3 cells infected with either an empty lentiviral vector (empty) or with the indicated doxycycline-inducible lentiviral YAP constructs, were plated on soft fibronectin-coated acrylamide hydrogels in the presence of doxycycline for 24 h and treated with medium (−CQ) or with CQ (+CQ) for the last 4 h. (E) Representative confocal images showing GFP-LC3 puncta (green). (F) Bars show accumulating GFP-LC3 puncta, and represent mean + SD from 4 independent experiments (*P < 0.0001 compared to −CQ empty infected, **P < 0.001 compared to −CQ YAP 5SA-infected cells; 2-way ANOVA). (G) Representative confocal images of MII-GFP-LC3 cells treated for 20 h with ethanol (EtOH) or 0.4 μM LatA. Cells were treated without (−CQ) or with CQ (+CQ) for the last 4 h; GFP-LC3 (green). (Scale bar, 20 μm.) (H) Quantification of GFP-LC3 puncta of MII-GFP-LC3 cells treated as in G. Bars represent mean + SD from 3 independent experiments (*P < 0.05 compared to −CQ EtOH-treated cells; 2-way ANOVA). (I) Representative confocal images of MII-GFP-LC3 cells treated for 20 h with DMSO or ML-7 (10 μM, 25 μM); GFP-LC3 puncta (green). (Scale bar, 20 μm.) (J) Quantification of GFP-LC3 puncta of MII-GFP-LC3 cells treated as in I. Bars represent mean + SD from 3 independent experiments (*P < 0.0001; **P < 0.05 compared to DMSO; 1-way ANOVA).

Fig. 4.

YAP/TAZ require efficient autophagy flux to sustain their biological responses. (A and B) Impairing autophagic flux inhibits anchorage-independent growth. Representative pictures (A) and quantification (B) of colonies formed by MDA-MB-231 cells transfected with control siRNA (siCo) or with siRNAs targeting either ATG7 (siATG7) or YAP/TAZ (siYAP/TAZ) and then plated in soft-agar conditions. For pharmacological inhibition of autophagy, MDA-MB-231 were plated for soft-agar assay and treated with medium (vehicle) or with 2 independent autophagy inhibitors (CQ: 25 μM; 3-MA: 10 μM). (Scale bar, 200 μm.) Data are presented as mean + SD of 3 independent experiments. (*P < 0.0001 compared to siCo; §P < 0.0001 compared to vehicle; 1-way ANOVA). (C and D) Impairing autophagic flux inhibits YAP/TAZ-induced mammosphere formation. TAZ S89A-overexpressing MII cells (TAZ S89A), either transfected with the indicated siRNAs or treated with the autophagy inhibitors, were tested for mammosphere formation. MII cells transduced with empty vector (empty) were used as negative control of mammosphere growth. Data are mean + SD of 3 independent experiments. (*P < 0.0001 compared to empty siCo; **P < 0.0001 compared to TAZ S89A siCo; §P < 0.0001 compared to TAZ S89A vehicle; 1-way ANOVA). (E) Immunoblot analysis for LC3 protein of lysates obtained from acinar cells (acini) and YAP-induced pancreatic organoids (yDuct) treated with normal medium (−CQ) or with CQ 50 μM (+CQ) for 4 h before harvesting. (F) Schematic of the experiment performed with acinar cells isolated from Atg7^fl/fl; tetO-YAP^S127A mice. Pancreatic acini were coinfected with an adenoviral vector expressing rtTA (Ad-rtTA), to allow a doxycyline-inducible YAP expression, in combination with either a Cre- (Ad-Cre) or GFP-encoding adenoviral vector (Ad-Gfp), to obtain Atg7^−/− or Atg7fl/fl cells, respectively. (G) Bright field images of pancreatic acini treated as indicated and seeded in collagen-I based hydrogels. Atg7fl/fl acini lacking exogenous YAP expression (e.g., Ad-rtTA + Ad-Gfp NO DOXY) were used as negative control of pancreatic reprogramming. (Scale bar, 50 μm.) (H) The ability to form ductal organoids was scored as percentage of acinar colonies converting to ductal structures. Data are mean + SD (n = 3 independent replicates) from 1 of 3 experiments, providing similar results (*P < 0.0001, lane 2 vs. lane 1; §P < 0.01 lane 3 vs. lane 2; 1-way ANOVA). (I and J) Pancreatic acini from R26-rtTA; tetO-YAPS127A mice seeded in collagen-I based hydrogels were cultured in the presence of DOXY, to induce the expression of the transgenic YAP, and treated either with medium (vehicle) or with 2 independent autophagy inhibitors CQ (25 μM) or 3-MA (10 μM). Acini lacking exogenous YAP expression (NO DOXY) were used as negative control of pancreatic reprogramming. See also SI Appendix, Fig. S2C for schematic of the experiment. Bright field images (I) and quantification (J) of yDuct organoids obtained upon YAP-dependent reprogramming of acinar cells treated as described above. (Scale bars, 50 μm.) Data are mean + SD of 3 independent experiments. (§P < 0.0001 compared to lane 1; *P < 0.0001 compared to lane 2; 1-way ANOVA). (K and L) FACS-purified LD cells were transduced with an rtTA-encoding lentivirus in combination with an empty vector (empty) or an inducible lentiviral YAP construct (YAP). Cells were cultured in presence of doxycycline and treated either with medium (vehicle) or 3-MA 10 μM (3-MA) as indicated. Representative images (K) and quantification (L) of yMaSC colonies formed by the indicated cells 15 d after plating in 5% Matrigel cultures. See also SI Appendix, Fig. S2D for representative FACS plots illustrating LD cells sorting procedure and SI Appendix, Fig. S2E for schematic of the experiment. Data are mean + SD of 2 independent experiments with 2 technical replicates each (*P < 0.0001, YAP compared to empty; §P < 0.0001, YAP+3MA compared to YAP; 1-way ANOVA). See SI Appendix, Methods for additional information on YAP-induced reprogramming experiments.

Fig. 5.

YAP/TAZ control autophagic flux through their direct target Armus. (A) MII-GFP-LC3, infected with an empty vector (empty) or with the indicated siRNA-insensitive doxycycline-inducible lentiviral YAP constructs, were transfected with control (siCo) or YAP/TAZ-targeting siRNAs (siYAP/TAZ). Cells were treated with doxycycline, harvested 48 h after siRNA transfection, and analyzed by RT-qPCR for Armus mRNA levels. Data were normalized to empty-infected cells transfected with siCo (black bar). (*P ≤ 0.0001 siCo + YAP WT compared to empty siCo; §P ≤ 0.0001 siYAP/TAZ + YAP WT compared to empty siYAP/TAZ; 2-way ANOVA). See also SI Appendix, Fig. S3 A and B. (B) Validation by ChIP-qPCR in MDA-MB-231 cells of the YAP/TAZ-binding site on the Armus-associated enhancer (see also SI Appendix, Fig. S3 C and D). CYR61 promoter is positive control, HBB promoter is negative control (control). Data from 3 replicates (mean + SD) are shown normalized to the percent input (1% of starting chromatin used as input). (C) Armus depletion impairs anchorage-independent growth. Quantification of colonies formed by MDA-MB-231 transfected with siCo or with 3 independent Armus siRNAs (siArmus #1, #2, and #3) plated in soft-agar assays. (*P < 0.0001 compared to siCo; 1-way ANOVA). See also SI Appendix, Fig. S3G for the validation of siRNAs. (D and E) Armus is required for YAP/TAZ-induced mammosphere formation. TAZ S89A-overexpressing MII cells (TAZ S89A) were transfected with the indicated siRNAs and tested for mammosphere formation. MII cells transduced with empty vector (empty) are negative control. (*P < 0.0001 TAZ S89A siCo compared to empty siCo; §P < 0.0001 TAZ S89A siArmus compared to TAZ S89A siCo; 1-way ANOVA). (F) Representative confocal images of MII-GFP-LC3 transfected as in C. The Insets (2×) show higher magnification of the GFP-LC3 puncta. (G) Quantification of GFP-LC3 puncta of MII-GFP-LC3 cells treated as in F, measured as area of GFP-LC3 puncta per cell. (*P < 0.001, **P < 0.05 compared to −CQ siCo; 2-tailed Student’s t test). (H and I) MII-GFP-LC3, infected with an empty vector (empty) or with a siRNA-insensitive doxycycline-inducible lentiviral Flag-tagged YAP constructs (YAP), were transfected with siCo, YAP/TAZ siRNAs mix (siYAP/TAZ), or Armus siRNA (siArmus), and treated with doxycycline. Representative confocal images (YAP in red) (H) and quantification (I) of GFP-LC3 puncta of MII-GFP-LC3 cells treated as above, measured as area of GFP-LC3 puncta per cell. (*P < 0.0001 compared to lane 1; §P < 0.0001 compared to lane 2; n.s., not significant compared to lane 3; 1-way ANOVA). (J) MII-GFP-LC3, infected with an empty vector (empty) or with a doxycycline-inducible vector encoding a siRNA-insensitive HA-tagged mouse Armus construct (Mm Armus, red), were transfected with the indicated siRNAs. (K) Quantification of GFP-LC3 puncta of MII-GFP-LC3 cells treated as in J. (*P < 0.01, **P < 0.001 compared to lane 1; §P < 0.001 compared to lane 2; §§P < 0.0001 compared to lane 3; 1-way ANOVA). (F–K) Cells were treated with medium (−CQ) or CQ 50 μM (+CQ) for the last 4 h and analyzed 48 h after siRNA transfection. (F, H, and J) GFP-LC3 (green); DAPI (blue) is a nuclear counterstain. (Scale bar, 20 μm.) (A, C, E, G, I, and K) Bars represent mean (n = 3) + SD.