β-catenin knockdown promotes NHERF1-mediated survival of colorectal cancer cells: implications for a double-targeted therapy

Abstract

Nuclear activated β-catenin plays a causative role in colorectal cancers (CRC) but remains an elusive therapeutic target. Using human CRC cells harboring different Wnt/β-catenin pathway mutations in APC/KRAS or β-catenin/KRAS genes, and both genetic and pharmacological knockdown approaches, we show that oncogenic β-catenin signaling negatively regulates the expression of NHERF1 (Na⁺/H⁺ exchanger 3 regulating factor 1), a PDZ-adaptor protein that is usually lost or downregulated in early dysplastic adenomas to exacerbate nuclear β-catenin activity. Chromatin immunoprecipitation (ChIP) assays demonstrated that β-catenin represses NHERF1 via TCF4 directly, while the association between TCF1 and the Nherf1 promoter increased upon β-catenin knockdown. To note, the occurrence of a cytostatic survival response in settings of single β-catenin-depleted CRC cells was abrogated by combining NHERF1 inhibition via small hairpin RNA (shRNA) or RS5517, a novel PDZ1-domain ligand of NHERF1 that prevented its ectopic nuclear entry. Mechanistically, dual NHERF1/β-catenin targeting promoted an autophagy-to-apoptosis switch consistent with the activation of Caspase-3, the cleavage of PARP and reduced levels of phospho-ERK1/2, Beclin-1, and Rab7 autophagic proteins compared with β-catenin knockdown alone. Collectively, our data unveil novel β-catenin/TCF-dependent mechanisms of CRC carcinogenesis, also offering preclinical proof of concept for combining β-catenin and NHERF1 pharmacological inhibitors as a mechanism-based strategy to augment apoptotic death of CRC cells refractory to current Wnt/β-catenin-targeted therapeutics.

Fig. 1

β-catenin represses NHERF1 expression through associating with TCF4. a Ls174T and DLD1 cells stably transfected with a doxycycline (Dox)-inducible small hairpin (sh)RNA for β-catenin (shβ-Cat) or a scramble shRNA (shCtr) as control were cultured without (−) or with (+) Dox for 5 days. Total cell lysates were then probed for β-catenin (β-Cat) and NHERF1 protein amounts. Tubulin served as a loading control. b Ls174Tshβ-Cat and DLD1shβ-Cat cells expressing a Dox-inducible shRNA for β-catenin were cultured without (−) or with (+) Dox for 1, 2, 3, or 5 days and then assessed by western blotting as indicated. c An equal amount of RNA (1 μg) of Ls174Tshβ-Cat and DLD1shβ-Cat cells cultured in the absence (−) or presence of Dox at the indicated time points, was analyzed by RT-PCR for assessing nherf1 and β-actin mRNA levels. The normalized nherf1expression versus β-actin mRNA at day 5 was quantified by using Image J analysis software for Windows. Data from three independent experiments were presented as means ± SEM (*p < 0.05). d ChIP of TCF1 and TCF4 in the Nherf1 promoter region in Ls174Tshβ-Cat and DLD1shβ-Cat cells cultured in the absence (−) or presence of Dox (+) for 5 days. qPCR was performed to quantify ChIP assay results. Enrichment was quantified relative to input controls. An anti-IgG antibody was used as a negative control. Results are represented as the average ± SD of three independent experiments (*p < 0.01)

Fig. 2

Ectopic cytoplasmic and nuclear overexpression of NHERF1 in β-catenin-depleted CRC adenocarcinoma cells. a Ls174Tshβ-Cat were cultured in the absence or presence of Dox (−Dox/+Dox) for 5 days, stained with primary antibodies for β-catenin (β-Cat) or NHERF1 and visualized with a fluorescent IgG-TRITC (for β-Cat) or IgG-FITC (for NHERF1) secondary antibodies by laser scanning confocal microscopy. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI). A merge of the two fluorescence signals and DAPI staining is also shown (Magnification, ×60). Each confocal image is representative of three independent experiments. b DLD1shβ-Cat cells were cultured in the absence or presence of 2 μg/mL of Dox (−Dox/+Dox) for 5 days and then fractionated to obtain purified extracts from either membrane, cytosol, and nucleus of cells, as described in Methods section. Equal amounts of each cell extract (100 μg) were separated by SDS-PAGE and probed with the indicated antibodies

Fig. 3

Small molecule inhibitors of β-catenin promote NHERF1 overexpression and mislocalization in CRC cells. a Subconfluent Ls174Tshβ-Cat were co-transfected with the luciferase reporter constructs TOPflash or FOPflash as reported in Methods section. At 24 h after transfection, cells were treated with vehicle alone (DMSO), FH535 (1 μM) or pyrvinium pamoate (150 nM) for additional 72 h. Luciferase activity was expressed as fold activation compared with carrier control cells. Values represent means ± SD of three representative experiments. b After 4 days of treatment with DMSO, FH535 (1 μM) or pyrvinium pamoate (150 nM), total cell lysates were probed for β-catenin (β-Cat), c-Myc and NHERF1 expression. Tubulin served as a loading control. c Ls174Tshβ-Cat cells exposed for 4 days to DMSO, FH535 (1 μM) or pyrvinium pamoate (150 nM) were analyzed by propidium iodide-staining and flow cytometric analysis for assaying cell cycle distribution, as described in Methods section. A representative dot plot of three replicate experiments shows the percentages of cells into different phases of the cell cycle for each indicated treatment. d Each confocal image is representative of three indipendent immunofluorescence analyses of NHERF1 by a rabbit polyclonal antibody visualized with a fluorescent IgG-FITC secondary antibody for the indicated treatments (left panels). DAPI staining (right panels) is also shown (Magnification, ×60)

Fig. 4

β-catenin-silenced CRC cells show a unique protein profile centred around autophagy and energy metabolism. Ls174T and DLD1 cells expressing a doxycycline (Dox)-inducible shRNA for β-catenin (Ls174Tshβ-Cat and DLD1shβ-Cat) were cultured without or with 2 μg/mL of Dox (−Dox/+Dox) for 1, 3, or 5 days. a A representative image of cells from one of the six fields captured from each well by using a camera attached to an inverted Olympus IX51 microscope is shown. b After 5 days of treatment without or with doxycycline (−Dox/+Dox), Ls174Tshβ-Cat cells were analyzed by propidium iodide-staining and flow cytometric analysis for assaying cell cycle distribution, as described in Methods section. A representative dot plot of three replicate experiments shows the percentages of cells into different phases of the cell cycle for each indicated treatment (left panels). Under the same experimental conditions, Ls174Tshβ-Cat cells were analyzed for the presence of MDC-positive autophagosomes, distinct dot-like structures trapped in acidic, membrane-rich organelles distributed in the cytoplasm or localizing in the perinuclear regions of CRC cells (Magnification, ×60). The numbers indicate the percentage of autophagy induction for each treatment condition. c After 5 days of treatment without or with doxycycline (−Dox/+Dox), cell cycle distribution of DLD1shβ-Cat cells was analyzed by propidium iodide-staining and flow cytometric analysis. A representative plot of three replicate experiments shows the percentages of cells into different phases of the cell cycle for each indicated treatment (left panels). DLD1shβ-Cat cells were analyzed for the presence of MDC-positive autophagosomes, as previously reported for Ls174Tshβ-Cat cells (Magnification, ×60). The numbers indicate the percentage of autophagy induction for each treatment condition. d Total cell lysates from Ls174Tshβ-Cat and DLD1shβ-Cat cultured without (−) or with (+) Dox for 5 days were assessed by western blotting with the indicated antibodies

Fig. 5

Combined shRNA-mediated downregulation of β-catenin and NHERF1-induces apoptosis in human CRC cells. Subconfluent Ls174T cells stably harboring a doxycycline (Dox)-inducible shRNA for β-catenin (Ls174Tshβ-Cat) were further transiently transfected with 200 nM of NHERF1 targeted shRNAs (shNHERF1) or scramble shRNAs as control (shCTR). a Total protein extracts from double shβ-Cat/shCTR or shβ-Cat/shNHERF1 expressing cells cultured without (−) or with (+) Dox for 5 days to simultaneously induce shRNAs for β-catenin were assessed by western blotting with the indicated antibodies. b A representative image of Dox-treated (+Dox) or untreated shCTR or shNHERF1 expressing Ls174Tshβ-Cat or DLD1shβ-Cat cells from one of the six fields captured from each well by using a camera attached to an inverted Olympus IX51 microscope is shown. c Ls174Tshβ-Cat or DLD1shβ-Cat cells expressing a scramble control shCTR or specific shRNAs for NHERF1 (shNHERF1) were cultured without (−) or with (+) Dox for 5 days and then labeled by Annexin V/PI (propidium iodide) staining for apoptosis evaluation by flow cytometry. Values represent the mean ± SD of three independent experiments. d Caspase-3 activity was also measured and data reported as fold induction over vehicle-treated control samples

Fig. 6

RS5517 is a new PDZ1-domain antagonist of NHERF1. a The chemical structure of RS5517 derivative. b Proposed binding mode for derivative RS5517 (purple) to the NHERF1 PDZ1 domain that is depicted as green cartoon. Residues involved in interactions were reported as pink stick. H-bonds were shown as yellow dot lines. c Binding of PDZ1 NHERF1 Y38W to the ligand Dansyl-NDSLL in the presence (filled circles) and in the absence (open circles) of 5 mM RS5517. Fluorescence data were recorded in the presence of 50 mM Na phosphate, pH 7.2, 300 mM NaCl, 5 mM DTT, 20% DMSO, at 25 °C. Lines are the best fir to a hyperbolic binding transition. It is evident that binding between PDZ1 NHERF1 Y38W to the ligand Dansyl-NDSLL is abolished in the presence of RS5517

Fig. 7

RS5517 synergizes with shRNA-mediated silencing of β-catenin in promoting CRC cell death. a Proliferation assays of Ls174Tshβ-Cat or DLD1shβ-Cat cells exposed to increasing concentrations of RS5517 (range 0.1–100 μM) or vehicle alone (−) in the absence or concomitant presence of Dox (−Dox/+Dox) for 5 days. Data were expressed as % of growth inhibition. b Ls174Tshβ-Cat or DLD1shβ-Cat cells were treated with 10 μM RS5517 in the absence or presence of Dox (−Dox/+Dox) for 5 days and then labelled by Annexin V-PE for apoptosis evaluation by flow cytometry. Values represent the mean ± SD of three independent experiments. c Ls174Tshβ-Cat or DLD1shβ-Cat cells were treated with 10 μM RS5517 (+) or DMSO as vehicle control (−) in the absence or concomitant presence of Dox (−Dox/+Dox) for 5 days and then analyzed by propidium iodide-staining and flow cytometric analysis for assaying cell cycle distribution, as described in Methods section. A representative dot plot of three replicate experiments shows the percentages of cells into different phases of the cell cycle for each indicated treatment. d Number of Ls174Tshβ-Cat or DLD1shβ-Cat colonies formed in soft agar containing DMSO or 10 μM RS5517 after 14 days. The values are presented as mean ± SD of three independent experiments (*P < 0.05). A representative cell colony image was captured for the indicated treatment condition by using a camera attached to an inverted Olympus IX51 microscope

Fig. 8

RS5517 prevents nuclear import of NHERF1 and shβ-catenin-mediated accumulation of phospho-ERK1/2 and RILP in CRC cells. a DLD1shβ-Cat cells treated with 10 μM RS5517 or DMSO in the absence or concomitant presence of Dox (−Dox/+Dox) for 5 days were stained using an anti-NHERF1 rabbit polyclonal primary antibody followed by an IgG-FITC secondary antibody and DAPI staining for each indicated treatment. Each confocal image is representative of three independent immunofluorescence experiments (Magnification, ×60). b Ls174Tshβ-Cat cells were treated with 10 μM RS5517 or DMSO in the absence or concomitant presence of 2 μg/mL of Dox (−Dox/+Dox) for 5 days and then fractionated to obtain purified extracts from either membrane, cytosol, and nucleus of cells, as described in Methods section. Equal amounts of each cell extract (100 μg) were separated by SDS-PAGE and probed with the indicated antibodies. c Ls174Tshβ-Cat cells were exposed to 10 μM RS5517 in the absence or concomitant presence of 2 μg/mL of Dox (−Dox/+Dox) for 5 days and total lysates were then analyzed by western blotting with the indicated antibodies

Fig. 9

RS5517 induces CRC cell death when combined to pharmacological inhibitors of β-catenin. a Ls174Tshβ-Cat cells were treated with 10 μM RS5517 as single agent or in combination with FH535 (1 μM) or pyrvinium pamoate (150 nM) for 4 days and a representative image of one of the six fields captured from each well by using a camera attached to an inverted Olympus IX51 microscope is shown. b Ls174Tshβ-Cat or DLD1shβ-Cat cells treated with 10 μM RS5517 alone or in combination with FH535 (1 μM) or Pyrvinium pamoate (150 nM) for 4 days were then labelled by Annexin V/propidium iodide staining for apoptosis evaluation by flow cytometry. Values represent the mean ± SD of three independent experiments (*P < 0.003). c Total lysates from Ls174Tshβ-Cat or DLD1shβ-Cat cells treated with 10 μM RS5517 alone or in combination with FH535 (1 μM) or Pyrvinium pamoate (150 nM) for 4 days were analyzed by western blotting with the indicated antibodies