The Phorbol Ester PMA Synergizes with GSK3 Inhibition and this Cooperation Requires Macropinocytosis and Lysosome Acidification (A-A’) Albino mbryos injected into the gastrula cavity (40 nl at 1 mM) with the macropinocytosis marker TMR-dextran 70kDa were fixed and bisected at gastrula stage 11. Note that dextran macropinocytosis is stronger in the dorsal part of the embryo (asterisk; dorsal blastopore indicated by arrowhead). (C) PMA increases the effects of LiCl in Wnt signaling in early embryos. qRT-PCR analysis at blastula stage 9.5 of the Wnt target genes Siamois and Xnr3 normalized for Ornithine decarboxylase (ODC) transcripts. Embryos received a single injection of 4 nl of LiCl (300 mM with or without PMA (500 nM). (D) Uninjected control embryo at stage 24. (E) LiCl (4nl 300 mM, 1 x ventral) dorsalized the embryo only slightly. (F) EIPA (1 mM, 1x ventral) alone did not produce a distinctive phenotype at this concentration. (G) The vacuolar ATPase (V-ATPase) inhibitor Bafilomycin A1 (Baf) (incubation with 5 μM for 7 minutes at 32 cells) ventralized embryos. (H) A single injection of PMA (500 nM) did not show any phenotypic effect. (I) PMA and LiCl strongly synergized, resulting in embryos with radial heads. (J) Coinjection of the EIPA macropinocytosis inhibitor blocked dorsalization caused by LiCl plus PMA. (K) Incubation with Baf lysosomal acidification inhibitor blocked dorsalization caused by LiCl plus PMA. Number of embryos or hemisections analyzed were as follows: A = 150, 100%; A’ = 160, 97% with fluorescence; five independent experiments; D = 70, 100%; E = 80; 98%; F = 75, 94%; G = 82, 92%; H = 76, 100%; I = 112, 98% J = 75, 93%; K = 85, 98%. Scale bars, 500 μm; Error bars denote SEM (n ≥ 3) (**P < 0.01). Also see Figure S1.

Order-of-addition Experiment Showing that Lysosomal Acidification at 32-cell Stage is Required for Early Wnt/β-catenin Activation (A) Untreated embryo. (B) Baf treatment (5 μM for 7 minutes) inhibited endogenous axis formation resulting in ventralized embryos lacking heads at early tailbud. (C) Embryos treated with Baf, washed, and then immersed in 300 mM LiCl an additional 7 minutes formed heads, resulting in dorsalized embryos with enlarged heads and short trunks. Note that all treatments were done at 32-cell stage, which is critical for the early Wnt signal. (D) LiCl treatment alone dorsalized embryos. (E) Quantitative RT-PCR (qPCR) for Wnt target genes Siamois and Xnr3 at blastula, confirming that the phenotypic effects are due to early activation of the Wnt pathway. (F) Untreated embryo. (G) Embryos incubated with LiCl at 32-cell were dorsalized. (H) Embryos first treated with LiCl and then subsequently with Baf resulted in ventralized embryos lacking heads. (I) Baf treatment caused ventralization and small heads. (J) Quantitative RT-PCR for Wnt target genes Siamois and Xnr3 showing that inhibiting V-ATPAse at the 32-cell stage blocks the effect of earlier LiCl treatment. The numbers of embryos analyzed were as follows: A = 115, 100% B = 124, 98% with phenotype; C = 132, 97%; D 99%; F = 132, 100%; G = 135, 99%; H = 126, 97%; I = 129, 98% (Scale bars, 500 μm.). Error bars denote SEM (n ≥ 3) (**P < 0.01).

Induction of Secondary Axes by the GSK3/β-catenin Pathway Requires Membrane Trafficking (A) Control embryo at early tailbud. (B) Embryo injected with DN-GSK3 (dominant-negative GSK3-β, 150 pg 1 x ventral) mRNA, showing double axes (arrowheads). (C) Injection of EIPA 1 x ventral alone showed no phenotypic effect (4 nl,1 mM). (D) Coinjection of DN-GSK3 and EIPA blocked double axis formation. (E) Uninjected control embryo. (F) Activation of Wnt signaling via injection of β-catenin mRNA (80 pg) induced complete twinned axes (arrowheads). (G) Embryo coinjected with β-catenin mRNA and DN-Rab7 (500 pg) showing that membrane trafficking is required for secondary axis formation. (H) Coinjection of β-catenin mRNA and the macropinocytosis inhibitor EIPA blocks axial duplication. The numbers of embryos analyzed were as follows: A = 62, 100%; B = 75, 94% with double axes; C = 76, 100%; D = 70; 98%; E = 70, 100%; F = 73, 98%; G= 69, 75%; H = 82, 97%; four independent experiments; (Scale bars, 500 μm.). Error bars denote SEM (n ≥ 3) (**P < 0.01).

Rac1 Inhibitor EHT1864 Blocks Wnt Signaling in Xenopus Embryos, and Macropinocytosis Is Required for Nuclear β-catenin Accumulation in CRC Cells (A) Uninjected control embryo. (B) Incubation of the Xenopus embryos with the Rac1 inhibitor EHT at 32-cell stage (7 min, 10 mM) resulted in a ventralized phenotype with a small head in the anterior (A, arrowhead) and expanded ventral structures in the posterior (P). Rac1 activity is required for macropinocytosis, see Figure S2). (C) qRT-PCR of gastrula stage embryos showing increased ventral markers Szl and Vent1 after Rac1 inhibition. (D) Control embryo. (E) Injection of Wnt8 mRNA (2 pg) induces complete duplicated axes (arrows). (F) Injected embryos with EHT (1 mM, 4 nl 1 x ventral) alone showed no phenotypic effect at this concentration. (G) EHT coinjected with Wnt8 mRNA blocked double axis formation. (H) qRT-PCR of Wnt target genes Siamois and Xnr3 at blastula confirming that Rac1 is required for early Wnt signaling. (I-I’) The colorectal cancer cell line SW480, in which the Wnt signaling pathway is activated by APC mutation, was positive for Rac1 and nuclear β-catenin immunostaining. (I’’) Merged panel including the DNA stain DAPI. (J-J’’) SW480 cells treated with the EIPA inhibitor (40 μM) had strongly decreased Rac1 and nuclear β-catenin levels, indicating a requirement for macropinocytosis. The numbers of embryos analyzed were as follows: A = 52, 100%; B = 47, 95% with ventralized small head phenotype; D = 58, 100%; E = 67; 97%; F = 64, 96%; G = 62, 97%; four independent experiments. Scale bars for embryos 500 μm; scale bars for immunofluorescence, 10 μm. Experiments represent biological replicates. Error bars denote SEM (n ≥ 3) (**P < 0.01). Also see Figure S2.

Degree of Human CRC Malignancy is Associated with Increased Macropinocytosis/MVB/lysosome markers and decreased GSK3 levels (A-A’) Normal human colon paraffin section stained with the macropinocytosis marker Pak1 and β-catenin, respectively. (A’’) Merged image with DAPI, a few cells colocalize both markers. (B-B’’) Pak1 and β-catenin levels are moderately increased at an early stage I CRC adenocarcinoma. (C’-C’’) Strong colocalization between Pak1 and β-catenin was observed in advanced stage IV CRC (inset). (D-D’’) Normal human colon section stained with the MVB marker CD63 and β-catenin; colocalization was not observed. (E-E’’) CD63 and β-catenin were stabilized in adenocarcinoma I, and moderate colocalization was found between CD63 and β-catenin. (F-F’) CD63 and β-catenin were strongly stabilized and colocalized in adenocarcinoma stage IV CRC. (F’’) Merge; note the striking colocalization between the MVB marker CD63 and β-Catenin in advanced stages of cancer (inset). (G-G’’) Normal colon stained for V0a3 (a subunit of V-ATPase that marks lysosomes) and β-catenin. (H-H’’) Stage I adenocarcinoma with moderately increased levels of lysosomes and β-catenin. (I-I’’) Strong co-localization of lysosomes and β-catenin in stage IV CRC (see inset). (J-J’’) Human colon array stained with the GSK3 and β-catenin. (K-K’’) GSK3 decreases, and β-catenin increases, at early stages of carcinogenesis, (L-L’’) GSK3 levels are very low in advanced CRC compared to normal human colon, while β-catenin levels are very high. Scale bars, 10 μm. Also see Figure S3 for quantifications and mouse xenografts of CRC cells.

A Focal Adhesion Protein Changes Its Nucleocytoplasmic Distribution after Wnt Signaling Activation, and FAK is Stabilized by PMA (A) Diagram showing how focal adhesions connect the actin cytoskeleton to the extracellular matrix via integrins; Wnt signaling changes the distribution of the focal adhesion protein Tes from focal adhesion sites to the nucleus. (B) Western blot of SW480 cells with and without stable expression of APC; note the reduction in Tes expression and increase in β-catenin levels in SW480 cells lacking full-length APC. GADPH was used as loading control. (C-C’) Colon cancer cell line SW480 stably restored with full-length APC stained with Tes and β-catenin antibodies; note that in SW480APC cells TES is absent from the nucleus (inset). (D-D’’) In SW480 CRC cells both Tes and β-catenin are located the nucleus. (E-E’’) Colon cancer cell line SW480 stained with Tes (in green) and β-catenin (in red) antibodies as a control. (F-F’’) Inhibiting macropinocytosis with EIPA (40 μM) treatment restored the cytoplasmic localization of the focal adhesion protein Tes in SW480 cells and strongly inhibited β-catenin levels. (G) Quantification of colocalization between Tes and β-catenin in normal mouse colon and SW480 xenografts. (H-H’) Immunohistochemistry of the CD1 NU/NU normal mouse colon showing Tes and β-catenin distribution. (H’’) Merge showing modest co-localization (inset). (I-I’’) Immunohistochemistry images of the CD1 NU/NU mouse xenograft model showing nuclear TES, which is strongly colocalized with β-catenin levels in cancer cells (inset). (J) Reporter assay in HEK293T cells stably expressing BAR and Renilla, showing strong inhibition of the β-catenin transcriptional activity induced by LiCl (40 mM) by FAK inhibitor PF-00562271 (20 μM) after overnight treatment. See also Figure S4A-S4B’’. (K-K’’) SW480 cells immunostained with FAK and β-catenin antibodies. (L-L’’) The tumor promoter PMA (750 nM, overnight treatment) increased levels of both β-catenin and FAK in SW480 cells (in which the Wnt pathway is constitutively activated by APC mutation). This increase in FAK levels requires membrane macropinocytosis and lysosome activity (see Figure S4C-S4H’’). Error bars denote SEM (n ≥ 3) (**P < 0.01) (Scale bars, 10 μm). Also see Figure S4.

Model of PMA Synergy with Wnt Signaling through Macropinocytosis and Membrane Trafficking Wnt and PMA are activators of macropinocytosis, which is a cell drinking process driven by an actin meshwork that requires the small GTPase Rac1. Membrane trafficking into MVBs/lysosomes (marked by CD63) requires acidification by V-ATPase, which is inhibited by Bafilomycin A (Baf). Phorbol esters such as PMA stimulate macropinocytosis and synergize with Wnt signaling. Inhibiting macropinocytosis with the membrane trafficking inhibitors EIPA, Baf, or the Rac1 inhibitor EHT1864 block Wnt signaling and its cooperation with the tumor promoter PMA.

In Cultured Cells, the Phorbol Ester PMA Cooperates with GSK3 Inhibition in β-catenin Signaling, and this Requires Lysosomal Acidification; Related to Figure 1 (A) HEK293BR (BAR/Renilla) cells stained with total β-catenin antibody. (B) HEK293BR cells treated with Baf (20 μM) overnight show no changes in β-catenin levels. (C) Treatment with PMA (1.5 μM) slightly increased β-catenin levels. (D) HEK293BR cells treated with PMA and Baf had low β-catenin levels. (E) Cells treated with LiCl (40 mM) showed an increase in β-catenin. (F) Baf treatment blocked the increase in β-catenin caused by LiCl. (G) PMA cooperated with LiCl in the induction of β-catenin levels. (H) Baf treatment blocked the cooperative effect of LiCl and PMA, indicating a requirement for lysosome acidification. (I) Quantification of β-catenin fluorescence per cell with the different treatments indicated above. (J) Luciferase assay in HEK293BR cells showing cooperation between PMA and LiCl, and its inhibition by Baf. All experiments with cultured cells were biological triplicates. Error bars denote SEM (n ≥ 3) (**P < 0.01). (Scale bar, 10 μm.).

Macropinocytosis is Inhibited in SW480 CRC Spheroids by Treatment with the Rac1 Inhibitor EHT; Related to Figure 4 (A) Control SW480 spheroids at 96 hours of inverted drop culture. (B) Treatment with 300 nM Rac1 inhibitor (EHT1864) reduced the diameter of SW480 spheroids at 96 hours. (C) Quantification of spheroid area after EHT treatment. (D) The same spheroids incubated with the macropinocytosis marker TMR-dextran 70kDa for 1 hour. (E) EHT treatment strongly inhibited macropinocytosis. (F) Quantification of macropinocytosis inhibition. Six spheroids were plated per condition, in triplicate. Error bars denote SEM (n ≥ 3) (**P < 0.01). (Scale bars, 500 μm.).

Malignancy of Colorectal Cancer Positively Correlates with Macropinocytosis, MVB, and Lysosome Markers, and Inversely Correlates with GSK3 Levels; Related to Figure 5 (A-D) Quantification of the colocalization between Pak1, CD63, V0a3, and GSK3 with β-catenin in normal colon and at different stages of cancer from the histological sections shown in Figure 5. (E-E’) Immunohistochemistry of normal colon tissue from the CD1 NU/NU nude mice stained for the macropinocytosis marker Pak1 and β-catenin. (E’’) Merge with DAPI. (F-F’’) SW480 xenograft showing correlation between high levels of Pak1 and β-catenin colocalization (see inset). (G) Quantification of colocalization of Pak1 and β-catenin. (H-H’’) Control colon immunostained for V0a3 and β-catenin. (I-I’’) SW480 tumor showing a strong increase in the lysosomal marker V0a3 and β-catenin. (K-K’) Normal colon sample from mouse stained for GSK3 and β-catenin staining. Note that normal colon has GSK3 staining. (L-L’’) SW480 xenograft tumor showing reduced levels of GSK3, and high levels of β-catenin. (M) Quantification of the levels of GSK3 in normal conditions and in tumor cells; note that the decrease in GSK3 levels supports the GSK3 sequestration model of Taelman et al. (2010). Error bars denote SEM (n ≥ 3) (**P < 0.01). (Scale bars, 10 μm). Human tissue arrays were quantfied in triplicate fields.

The Phorbol Ester PMA Stabilizes FAK and enhances β-catenin levels in cells with constitutive Wnt signaling; Related to Figure 6 (A-A’’) SW480 cells immunostained for FAK and total β-catenin antibody. (B-B’’) FAK inhibitor (100 nM) reduces FAK and β-catenin immunostaining levels. (C-D’’) SW480 cells treated with PMA showed increased in β-catenin and FAK levels. These images are the same as those in Figure 5K-L’’, reproduced here as controls for the effects of membrane trafficking inhibitors in the same experiment. (E-E’’) EIPA (40 μM) overnight treatment blocked the effect of PMA in SW480 cells. (F-F’’) Baf (500 nM) treatment also blocked the effect of PMA in CRC cells. (G-G’’) EIPA treatment alone was sufficient to reduce β-catenin and FAK levels in SW480 cells. (H-H’’) Baf treatment in SW480 reduces β-catenin and FAK levels in SW480 cells. Experiments in cultured cells represent biological triplicates. Error bars denote SEM (n ≥ 3) (**P < 0.01). (Scale bars, 10 μm.).