KRAS Promotes Intestinal Cell Proliferation and Nuclear Localization of β-Catenin following Loss of APC

(A) apc^mcr zebrafish embryos were injected at the one-cell stage with KRAS mRNA. The 72 hpf embryos were fixed, sectioned, and stained for hematoxylin and eosin (H&E) (row 1, sagital section; row 2, cross section), DNA (magenta, middle and bottom), and either PCNA (middle, green) or β-catenin (bottom, green). Overlapping expression is shown in white.

(B) Protein lysates from 72 hpf embryos were subjected to western blot analysis for either V5-tag (top-left) and total RAS (bottom-left) or phospho-Erk (top-right) and vinculin (bottom-right).

(C) Zebrafish harboring an integrated β-catenin TOPGFP reporter were injected with KRAS mRNA, APC morpholino, or both. The 72 hpf embryos were subjected to whole-mount in situ hybridization for GFP. Boxes indicate the intestine (top), and arrows indicate the hindbrain (bottom).

All images were captured using the same exposure and represent at least three independent experiments. Scale bar, 10 μm.

KRAS and RAF1 Are Necessary for Nuclear Localization of β-Catenin and Intestinal Cell Proliferation following Loss of APC

(A) apc^mcr zebrafish embryos injected with constitutively active RAF1 or MEK1. The 72 hpf embryos were stained for hematoxylin and eosin (H&E) staining (top), DNA (magenta, middle and bottom), and either PCNA (middle, green) or β-catenin (bottom, green).

(B) SW-480 cells transfected with control, KRAS^G12V, RAF1-directed siRNA, or DN-MEK1 constructs were stained for DNA (magenta) and β-catenin (green).

(C) RNA was harvested and subjected to rt-PCR for Axin2. Error bars, SEM.

(D) SW-480 cells transfected with KRAS^G12V-specific siRNA were cotransfected with constitutively active KRAS, RAF1, or MEK1 and stained for DNA (magenta) and β-catenin (green).

Overlapping expression is shown in white. All images were captured using the same exposure and represent three independent experiments. Scale bar, 10 μm.

KRAS and RAF1 Direct Stabilized β-Catenin to the Nucleus

(A) WT and KRAS-injected zebrafish embryos treated with PGE₂ were stained for DNA (magenta), PCNA (green), and β-catenin (green).

(B) Human 293 cells were transfected with constitutively active KRAS, RAF1, or MEK1 and either DMSO (top) or PGE₂ (bottom). Cells were stained for DNA (magenta) and β-catenin (green).

(C) 293 cells treated with DMSO, PGE₂ or PGE₂, and leptomycin B were stained for DNA (magenta) and β-catenin (green).

(D) 293 cells were transfected with control or KRAS- or RAF1-directed siRNA and treated with EGF and PGE₂ or vehicle and then stained for DNA (magenta) and β-catenin (green).

(E) 293 cells were subjected to an MTT assay (^*p < 0.05 versus DMSO; error bars, SEM).

Overlapping expression is shown in white. All images were captured using the same exposure and represent three independent experiments. Scale bar, 10 μm.

KRAS/RAF1 Regulation of β-Catenin Requires the Activity of RAC1

(A and B) SW-480 cells transfected with vehicle or myc-tagged ([A], S191A or S605A) or flag-tagged ([B], S552A or S675A) β-catenin mutants were stained for DNA (magenta) and α-myc ([A], green) or α-flag ([B], green).

(C) SW-480 cells were transfected with RAC1-directed siRNA, DN-RAC1, and DN-Cdc42 or treated with the RAC1-specific inhibitor NSC23766 and stained for DNA (magenta) and β-catenin (green).

(D) SW-480 cells were subjected to an MTT assay (^*p < 0.05 versus DMSO; error bars, SEM).

(E) SW-480 cells were transfected with vehicle, KRAS^G12V-targeted siRNA, RAF1 siRNA, or DN-MEK1 and subjected to a RAC1 activity assay. The western blot was probed for RAC1 (top). Control lysates were probed for total RAC1 (bottom).

(F) SW-480 cells treated as above were stained for phospho-cJun.

(G) Human 293 cells transfected with constitutively active RAC1 and treated with PGE₂ or DMSO were stained for DNA (magenta) and β-catenin (green).

All images were captured using the same exposure and are representative of at least three independent experiments. Scale bar, 10 μm.

KRAS-Mediated Intestinal Cell Proliferation following Loss of APC Requires β-Catenin

(A) Zebrafish embryos were injected with β-catenin^S45A mRNA (left) along with KRAS mRNA (middle). Also shown is a representative image of the APC-KRAS embryo (right). At 72 hpf, the embryos were fixed and photographed.

(B) The percent cyclops was analyzed (^*p < 0.01 WT versus β-catenin-KRAS, ^**p < 0.01 WT versus APC-KRAS).

(C) Protein was harvested from apc^mcr embryos treated with DMSO or NS-398 and subjected to western blot analysis for β-catenin (top) or vinculin (bottom).

(D) Wild-type uninjected or apc^mcr embryos injected with KRAS mRNA treated with VEH (top) or NS-398 (bottom) were stained by H&E (WT, left; APC-KRAS, right) and for DNA (magenta), PCNA (green), and β-catenin (green). TOPGFP-APC^mo-KRAS embryos were stained for GFP expression (purple). Boxes indicate the intestine.

(E) SW-480 cells treated with DMSO or NS-398 were stained for DNA (magenta) and β-catenin (green). Protein lysates were subjected to western blot analysis for β-catenin (top) and vinculin (bottom).

(F) Cells were subjected to MTT analysis (^*p < 0.05 versus DMSO; error bars, SEM).

Overlapping expression is shown in white. All images were captured using the same exposure and represent at least three independent experiments. Scale bar, 10 μm.

apc Control of Cellular Fate and Differentiation Are Mediated by ctbp1 and Are β-Catenin Independent

(A) Protein from 48 hpf apc^mcr zebrafish embryos injected with either 6 x His-APC^955–2075 or 6 x His-APC^955–2075-AALP were subjected to western blot analysis for β-catenin (top), ctbp1 (second), 6 x His (third), or vinculin (bottom).

(B) Embryos injected as above were fixed at 72 hpf and subjected to in situ hybridization for i-fabp.

(C) apc^mcr embryos were treated with DMSO or NS-398 or injected with ctbp1-directed morpholino in the presence or absence of NS-398. At 72 hpf, the embryos were fixed and subjected to in situ hybridization for i-fabp, NaPi, hoxa13a, or evx1.

All images are representative of at least three independent experiments.

JNK1 Activation Is Coincident with Nuclear Accumulation of β-Catenin in Human Tumor Samples

FFPE human-matched grossly uninvolved and adenoma samples obtained from FAP patients and unmatched sporadic carcinomas were stained to indicate DNA (magenta) and (A) CtBP (green), (B) β-catenin^* (green), or (C) phospho-cJun (green) as an indicator of JNK activity. Overall, 20 patients matched grossly uninvolved, and adenoma or unmatched carcinoma tissue samples were stained. Shown are two representative samples. All images were captured using the same exposure, and overlapping expression is shown in white.

^*Note: A section from each of the β-catenin-stained adenomas was enlarged to the right. Each antibody was evaluated using serial sections. Scale bar, 5 μm.

KRAS Promotes Intestinal Cell Proliferation and Activation of β- Catenin following Loss of apc
(A) apc^mcr zebrafish embryos were injected at the one cell stage with KRAS mRNA. The embryos were fixed at 72 hpf, sectioned and stained using hematoxylin and eosin (H&E). Shown are representative sagital sections. (B) Zebrafish embryos harboring an integrated β-catenin TOPdGFP reporter were injected with KRAS^G12D mRNA, apc morpholino or both. The embryos were fixed at 72 hpf and subjected to whole mount in situ hybridization using GFP as a probe. (C) mRNA was harvested from 72hpf embryos injected as in (B). PCR was performed for GAPDH and unspliced apc. All images represent at least three independent experiments. (Scale Bar: 10μm).

APC-KRAS and APC-RAF1 Share Morphologic Abnormalities that Are Distinct from APC-MEK1 Embryos
(A) apcmcr zebrafish embryos were injected with constitutively active KRAS, RAF1 or MEK1. Protein lysate from 72 hpf embryos was harvested and subjected to western blot analysis for phosphor-Erk (top) and vinculin (bottom). (B) Embryos injected as above were fixed and photographed at 72 hpf. All images represent at least three independent experiments.

KRAS/RAF1 and COX-2/PGE₂ Are Required to Maintain Nuclear Localization of β-Catenin in Human Cells
(A) SW-480 cells were transfected with control, KRAS^G12V- or RAF1-direceted siRNA or a dominant negative MEK1 construct. Whole cell protein was harvested and subjected to western blot analysis for total RAS and vinculin (top), total RAF and vinculin (middle) or pERK and vinculin (bottom). (B) SW-480 cells were transfected with KRAS or RAF1-directed siRNA constructs. Two days after transfection, the cells were fixed and stained for β-catenin (green) or ToPro3 (magenta) or whole cell lysates were harvested and subjected to western blot analysis for total RAS (top – left), total RAF1 (top – right) or vinculin (bottom). (C) SW-480 cells were transfected with KRAS^G12V-directed siRNA and transfected with constitutively active V5-KRAS, 6xHis-RAF1 or MEK1-ERK2 fusion. Whole cell lysates were harvested 48 hours after transfection and subjected to western blot analysis for V5 (top), total RAS (middle) and vinculin (bottom), 6xHis (top), total RAF1 (middle) and vinculin (bottom), or MEK1 (top) and vinculin (bottom). (D) SW-480 cells were treated with a dose curve (0.1μM, 0.5μM, 1μM or 5μM) of the MEK-specific inhibitor U0126. Following 6 hrs of treatment, whole cell protein and nuclear fractions were harvested and subjected to western blot analysis for whole cell pERK (top) and vinculin (2^nd row) or nuclear β- catenin (3^rd row) and histone H3 (bottom). (E) 293 cells transfected with VEH, constitutively active, KRAS, RAF1 or MEK1 and treated for 6 hrs with PGE₂. Subcellular fractionation was performed and the fractions were subjected to western blot analysis for β-catenin (nuclear-top) or histone H3 or β-catenin (cytoplasmic-bottom) and cytochrome C. (F) 293 cells were treated with both PGE₂ and EGF for 0, 0.25, 0.50, 4 or 18 hrs. Following treatment, subcellular fractionation was performed and the fractions were subjected to western blot analysis for β-catenin (nuclear-top) or histone H3 or β-catenin (cytoplasmic-bottom) and cytochrome C. Total β-catenin was also assayed from whole cell lysate. All images are representative of at least three individual experiments.

RAC1, but Not Cdc42, Activity Is Necessary for Nuclear Localization of β-Catenin in SW-480 Cells
(A) SW-480 cells were transfected with a RAC1-directed siRNA. Whole cell lysate were harvested and subjected to western blot analysis for total RAC1 and vinculin (top). Subcellular fractionation was performed on cells treated as above and the nuclear fraction was subjected to western blot analysis for β-catenin and histone H3 (bottom). (B) Sw-480 cells were transfected myc-tagged dominant negative Cdc42 construct. Whole cell lysate was collected and subjected to western blot analysis for myc and vinculin. Also, a Cdc42 activity assay was performed. (C) SW-480 cells were transfected with a RAC1-directed siRNA construct. Two days after transfection, the cells were fixed and stained for β- catenin (green) or ToPro3 (magenta) or whole cell lysates were harvested and subjected to western blot analysis for total RAC1 (top) or vinculin (bottom). All images are representative of at least three independent experiments.

ctbp1 Knockdown Does Not Affect β-Catenin Levels
(A) apc^mcr zebrafish embryos were injected at the one-cell stage with ctbp1 morpholino 1. At 48 hpf, whole embryo protein was harvested and subjected to western blot analysis for ctbp1 (top), β-catenin (middle) and vinculin (bottom). (B) apc^mcr zebrafish embryos were injected at the one-cell stage with ctbp1 morpholino 2. At 72 hpf, the embryos were fixed and subjected to in situ hybridization for i-fabp, NaPi, hoxa13a and evx1. (C) At 48 hpf, whole embryo protein was harvested from embryos injected as in (B) and subjected to western blot analysis for ctbp1 (top) and vinculin (bottom). The images are representative of three individual experiments.