Journal: Frontiers in Oncology

Article Title: Chemotherapy induces feedback up-regulation of CD44v6 in colorectal cancer initiating cells through β -catenin/MDR1 signaling to sustain chemoresistance

doi: 10.3389/fonc.2022.906260

Figure Lengend Snippet: Both palmitoylation and linkage to a nuclear localization site of CD44v6 contribute to recruitment of LRP6 to caveolin1-rafts to regulate CD44v6-induced WNT3A/ β -catenin signaling. (A) , Schematic representations of individual CD44v6 mutants are shown; ED, extracellular domain; TM, transmembrane domain; ICD, intracellular domain. (B) , CD44v6 negative SW480-FR/(NON-CICs) were transfected with individual CD44v6 mutants as depicted. Individual CD44v6 cell clones were either untreated (control) or challenged with 1 x FOLFOX for 30 minutes. Raft (R) and non raft (NR) fractions were prepared as described in Methods. (C) , SW480-S and SW480-R cells were incubated with biotin conjugated anti-CD44v6 antibody at 4°C separately followed by further incubation at 37°C for 10, 20 and 30 minutes as indicated. The percentage of internalization was measured by flow cytometry after staining with fluorescein conjugated anti-biotin antibody. Data were calculated by setting the mean fluorescence intensity of cells after biotin labeling without glutathione incubation as 100%. (D) , SW480-FR cells were cultured in complete media with and without K+ depletion at 37°C for 1 hour followed by further stimulation with WNT3A for 30 minutes. Total cell lysates and endosomes purified by sucrose gradient centrifugation were analyzed by western blotting. (E-F) , SW480-FR cells were surface labeled with biotinylating agent (non-cleavable Sulfo-NHS-LC-Biotin). Cells were stimulated with WNT3A at 37°C for the times indicated and placed at 4°C for 1 hour of labelling with the biotinylating agent. Cytosolic membrane and nuclear membrane fractions were affinity purified with avidin-conjugated beads and analyzed by western blotting. (G-I) , Sensitive and FR SW480 cells were stably transfected with vector or a DAB2 construct. These stable clones were co-transfected with NTshRNA1, or caveolin1 (CAV1) shRNA1, or clathrin shRNA1. After 48 hours, cells were then transfected with TOP/FOPFlash luciferase reporter constructs prior to 20 ng/ml WNT3A stimulation for 12 hours, and cell lysates were subjected to luciferase activity determination (G) and processed for WB analysis for the indicated proteins (H-I) . (J-K) , Validations of CAV1shRNAs (CAV1 sh1 and CAV1 sh2) and Clathrin shRNAs (Clathrin sh1 and Clathrin sh2) were done by the indicated shRNA mediated knockdown and the corresponding knock-in (KI) gene transfections as described in Methods. Target proteins were analyzed by WB analysis ( β -tubulin, internal control). FACS data in ‘C’ represent are representative of 4 independent experiments. All WBs are representative of 3 independent experiments. QPCR data represent results from 3 independent experiments done in n = 3-6 replicates. (C) , * P < 0.05 was considered significant, Internalization of SW480-FR cells was compared with SW480-S cells. Data in ‘G’ represent results from 3 independent experiments performed in triplicates; * P < 0.05 was considered significant, TOPFlash/FOPFlash activity of CAV1 shRNA1 transfectant results were compared with NT shRNA transfectant of SW480-FR-Vector transfectant cells; TOPFlash/FOPFlash activity of clathrin shRNA1 transfectant results were compared with NT shRNA transfectant of SW480-FR-DAB2 cells.

Article Snippet: The M50 Super 8x TOPFlash vector (plasmid 12456) with a luciferase gene under the control of seven TCF/LEF-binding sites and the corresponding M51 Super 8x FOPFlash vector (plasmid 12457) with mutated TCF/LEF-binding sites were obtained from Addgene (Cambridge, MA, USA).

Techniques: Transfection, Clone Assay, Control, Incubation, Flow Cytometry, Staining, Fluorescence, Labeling, Cell Culture, Purification, Gradient Centrifugation, Western Blot, Membrane, Affinity Purification, Avidin-Biotin Assay, Stable Transfection, Plasmid Preparation, Construct, Luciferase, Activity Assay, shRNA, Knockdown, Knock-In

Journal: Frontiers in Oncology

Article Title: Chemotherapy induces feedback up-regulation of CD44v6 in colorectal cancer initiating cells through β -catenin/MDR1 signaling to sustain chemoresistance

doi: 10.3389/fonc.2022.906260

Figure Lengend Snippet: Nuclear localization site (NLS) in the ICD domain of CD44v6 is required for nuclear translocation of CD44v6 through endosomal sorting, and its subsequent association with TCF4 contributes to enrichment of TCF4/TOPFlash transcription. (A) , Associations of CD44v6 with LRP6 and actin were examined in pooled lipid raft fractions isolated from SW480-FR NON-CICs/CD44v6 cell clones expressing the indicated CD44v6 mutants (see the structures of CD44v6 mutants in Figure 9A ). After stimulation with WNT3A for 12 hours, the cell lysates from the individual CD44v6-expressing SW480-FR NON-CIC clones were immunoprecipitated (IP) with an anti-CD44v6 antibody, followed by fractionation and western blotting. (B) , WB analyses are shown for endosomal and nuclear fractions in individual SW480-FR NON-CICs/CD44v6 cell clones expressing the v6 Δ67 mutant (devoid of ICD) and v6 NLS mutants (devoid of nuclear localization site; see figure 9A ). (C) , Nuclear (N) and cytosolic (C) fractions were immunoprecipitated with TCF4 or IgG (Control) followed by Western blotting for the CD44v6, β -catenin, MDR1 and TCF4 proteins in the SW480-FR cells, and in the COS-7-CD44v6 clones expressing the indicated mutants and vector controls. (D) , Nuclear extracts were prepared from the parental HT29-FR, LOVO-FR, and SW480-FR cells, or from cell clones stably harboring lentivirus-encoded NT sh1, or v6 sh1, and they were immunoprecipitated with TCF4 antibody followed by Western blotting with the indicated antibodies. Whole cell lysates (WCL) from the same experiment were used as input and subjected to WB analysis for CD44v6. (E) , SW480-FR NON-CICs/CD44v6 cells were incubated with biotin-conjugated CD44v6 at 4°C for 1 hour followed by an additional hour of incubation at 37°C. Cytosolic and nuclear fractions were isolated and immunoprecipitated with streptavidin beads and analyzed by Western Blotting. (F) , Lysates from indicated SW480-FR-NON-CIC/CD44v6 cell clones expressing the indicated CD44v6 mutants were subjected to cytosol and membrane fractionation and then analyzed by WBs. The relative purities of the membrane and cytosolic fractions were confirmed by probing for the cytoplasmic protein HSP90 and the membrane protein transferrin receptor (Tf-R). (G–H) , SW480-FR-NON-CIC/CD44v6 cell clones expressing the pCD44v6/Δ67mutant (G) , and the pCD44v6/NLS mutant (H) , were transfected with TOPFlash and control TK-Renilla vectors, or with FOPFlash and TK-Renilla vectors in the presence or absence of 20 ng/ml of WNT3A. After 48 hours, cells were lysed and subjected to luciferase measurements and in parallel to WB analysis. All WBs are representative of 4 independent experiments. All luciferase data represent at least 3 independent experiments done in triplicates. (G, H) , * P < 0.05 was considered significant, TOPFlash/FOPFlash activity of WNT3A treated SW480-FR/NON-CICs/CD44v6 Δ67 Mut cells were compared with SW480-FR/NON-CICs/CD44v6 cells, and TOPFlash/FOPFlash activity of WNT3A treated SW480-FR/NON-CICs/CD44v6 NLS Mutant was compared with SW480-FR/NON-CICs/CD44v6 cells.

Article Snippet: The M50 Super 8x TOPFlash vector (plasmid 12456) with a luciferase gene under the control of seven TCF/LEF-binding sites and the corresponding M51 Super 8x FOPFlash vector (plasmid 12457) with mutated TCF/LEF-binding sites were obtained from Addgene (Cambridge, MA, USA).

Techniques: Translocation Assay, Isolation, Clone Assay, Expressing, Immunoprecipitation, Fractionation, Western Blot, Mutagenesis, Control, Plasmid Preparation, Stable Transfection, Incubation, Membrane, Transfection, Luciferase, Activity Assay

Journal: Frontiers in Oncology

Article Title: Chemotherapy induces feedback up-regulation of CD44v6 in colorectal cancer initiating cells through β -catenin/MDR1 signaling to sustain chemoresistance

doi: 10.3389/fonc.2022.906260

Figure Lengend Snippet: CICs isolated from resistant cells demonstrate resistance to FOLFOX treatment through WNT3A/ β -catenin signaling. (A) , QPCR (Upper panel) and western blot (Lower Panel) data are shown for CD44v6 mRNA (Upper panel) and protein expression (Lower panel) in CICs isolated from SQ tumors of SW480-FR, SW480-OXA, SW-5-FU, and SW480-S cells that were treated with or without either 1 x FOLFOX for 12 hours, Inset: CICs isolated from SQ tumors of SW480-FR, SW480-OXA, SW-5-FU, and SW480-S cells that were treated with either 1 x FOLFOX, or 1.2 ng/ml of WNT inhibitor LGK974 (IC 50, for LGK974 in SW480-S and SW480-FR cells are 0.8 ng/ml and 1.15 ng/ml [data not shown]), or 1 x FOLFOX + LGK974 for 12 hours. QPCR analysis was done with total RNA extracted from these treated cells and data are shown for CD44v6 mRNA. (B) , Secretion of WNT3A was measured by ELISA in sensitive and FR cells of SW480 after treatment with DMSO. Or 1 x FOLFOX, or 1.2ng/ml of LGK974, or 1 x FOLFOX + LGK974 for the indicated times. (C) , Sensitive and FR cells of SW480 were transfected with 50 ng TOPFlash and 50 ng TK-Renilla vectors, or with 50 ng FOPFlash and 50 ng TK-Renilla vectors. The TOPFlash/FOPFlash promoter was activated by treatment with FOLFOX (1x) for 12 hours. Cells were lysed and subjected to luciferase measurements. (D-E) , Validations of β -catenin shRNAs ( β -catenin sh1 and β -catenin sh2) (D) and of constitutively active β -actin (E) used in the following experiments (H-K) were examined. In “D”, the indicated shRNA mediated knockdown and the corresponding knock-in (KI) gene transfections were dune as described in Methods. Target proteins were analyzed by WB analysis ( β -tubulin, internal control). (F) , CD44v6 negative PD-FR/NON-CICs were transfected with either TOPFlash and control TK-Renilla vectors, or with FOPFlash and TK-Renilla vectors together with increasing time of incubation with CD44v6 cDNAs. After 48 hours, the cells were stimulated with or without 20 ng/ml WNT3A for the indicated times. Then the cells were lysed and subjected to luciferase measurements (upper panel) or, in parallel, to WB analysis for CD44v6 and Flag. (G) , PD-FR CICs were transfected with NT sh1, or with CD44v6 sh1. After 48 hours, cells were analyzed for WNT3A stimulated β -catenin/TCF4 promoter luciferase activity as shown in upper panel or, in parallel, to WB analysis with the indicated proteins (lower panel). (H, J) , SW480-FR CICs were transfected with NT sh1, or CD44v6 sh1 (v6 sh1), or β -catenin sh1, or treated with DMSO, or 1.2 ng/ml of LGK974. 48 hours after the transfections, and 12 hours after the LGK974 treatment, cell growth was assessed by counting colonies in a clonogenic growth assay (H) , and apoptosis was assessed by the Annexin V positive stain assay (J) . (I, K) , SW480-FR Non-CICs were transfected with vector control, v6 cDNA, or CA β -catenin cDNA. 48 hours after the transfections, cell growth was assessed by clonogenic growth assay (I) , and apoptosis was assessed by the Annexin V positive stain assay (K) . Data are presented as Mean ± SD from n = 3-4 replicates in three independent experiments. All WB data are representative of 4 independent experiments. (A) * P < 0.05 was considered significant for red asterisks, CD44v6 mRNA levels of FOLFOX treated cells were compared with the DMSO treated cells; * P < 0.05 considered significant for the green and blue asterisks, CD44v6 mRNA levels of 1.2 ng/ml, LGK974 and FOLFOX + LGK974 treated cells were compared with DMSO, or FOLFOX treated controls. (B) , *P < 0.05, was considered significant, secreted WNT3A in LGK974 treated cells of SW480-S and SW480-FR were compared with their respective DMSO treated controls. (C) , *P < 0.05, was considered significant, FOLFOX treated cells of SW480-S and SW480-FR were compared with their respective DMSO treated controls. (D-E) , *P < 0.05, was considered significant, WNT3A treated PD-FR NON-CICs (D) and PD-FR CICs (E) at various time points were compared with their respective untreated controls. (F, H) , *P < 0.05, was considered significant, v6 shRNA1, β -catenin shRNA1, and LGK974 treated clonogenic growth (F) , and Annexin V positive (H) CICs were compared with their appropriate vector + NTshRNA, and DMSO controls. (G, I) , *P < 0.05, was considered significant, v6 cDNA, CA- β -catenin CDNA overexpressed clonogenic growth (G) , and Annexin V positive (I) NON-CICs were compared with their appropriate vector controls.

Article Snippet: The M50 Super 8x TOPFlash vector (plasmid 12456) with a luciferase gene under the control of seven TCF/LEF-binding sites and the corresponding M51 Super 8x FOPFlash vector (plasmid 12457) with mutated TCF/LEF-binding sites were obtained from Addgene (Cambridge, MA, USA).

Techniques: Isolation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Luciferase, shRNA, Knockdown, Knock-In, Control, Incubation, Activity Assay, Growth Assay, Staining, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: Chemotherapy induces feedback up-regulation of CD44v6 in colorectal cancer initiating cells through β -catenin/MDR1 signaling to sustain chemoresistance

doi: 10.3389/fonc.2022.906260

Figure Lengend Snippet: CD44v6 regulated β -catenin signaling establishes FOLFOX resistance in CRC-CICs. (A) , CD44 negative COS7 cells were stably transfected with vector control or with Flag-CD44v6 cDNA. Nuclear (N) and cytosolic (C) fractions were prepared from COS-7/Flag-tagged vector and COS-7/Flag tagged-CD44v6 stable transfectants and immunoprecipitated by the anti-Flag antibody. Flag-immunoprecipitated proteins were analyzed by Western blotting with the indicated antibodies. Upper panel of inset - Western blots of wild-type and Flag-tagged CD44v6 in Flag-CD44v6 knock-in clones and the parental clone (wild type CD44v6 transfectants of COS-7 cells) with either anti-CD44v6 or anti-Flag antibodies, Lower panel of inset - Cell lysates of CD44v6 × Flag knock-in and parental clones were immunoprecipitated with anti-Flag and western blotted with anti-CD44v6. (B-E) , CICs from SW480-S cells (B, D) and SW480-FR cells (C, E) were transfected with NT sh1 or v6 sh1. After 48 hours, CICs were analyzed for FOLFOX stimulated β -catenin/TCF4 promoter luciferase activity (B, C) as described in Figure 5C or, in parallel, to WB analysis with the indicated proteins (D, E) . (F, G) , CICs from SW480-S (F) and SW480-FR (G) cells were transfected with NT sh1 or CD44v6 sh1 vectors. After 48 hours, CICs were subjected to WB analysis for the indicated proteins. (H–J) , COS-7/vector and COS-7/Flag-CD44v6 stable clones were further transfected with 50 ng TOPFlash and 50 ng TK-Renilla vectors, or with 50 ng FOPFlash and 50 ng TK-Renilla vectors for 48 hours. They were then treated either with vehicle (DMSO) or with the MEK inhibitor U0126 (20 µM) (H) , or with the PI3K-Inhibitor Ly294002 (50 µM) (I) , or a casein kinase 1 inhibitor CKI-7 (hydrochloride) (CKI-7) (2 µM) (J) 2 hours prior to the addition of WNT3A. After 12 hours of induction with WNT3A, cells were lysed and subjected to luciferase measurements. (K) , PD-FR CICs were transfected with NT sh1 or v6 sh1. After 48 hours, cells were transfected with TOPFlash and TK-Renilla or with FOPFlash and TK-Renilla vectors. The TOPFlash/FOPFlash promoter was activated by stimulation with WNT3A (20 ng/ml) for 12 hours or by further transfection with LRP6, DVL2 or constitutively active (CA) β -catenin for 48 hours. Cells were lysed and subjected to luciferase measurements, and the data are presented as TOPFlash/FOPFlash promoter activity or, in parallel, to WB analysis. (L) , COS7-Vector and COS7-CD44v6 clones were transfected with TOPFlash and TK-Renilla vectors, or with FOPFlash and TK-Renilla vectors for 48 hours. The reporter was stimulated with 20 ng/ml WNT3A for 12 hours or by further transfection with LRP6, DVL2, or constitutively active (CA) β -catenin for 48 hours. Cells were lysed and subjected to luciferase measurements or, in parallel, to WB analysis. Data are presented as Mean ± SD from n = 3-6 replicates in three independent experiments. All WBs data are representative of 4 independent experiments. (B, C) , * P < 0.05 was considered significant, TOPFlash/FOPFlash activity in v6shRNA1 (v6sh1) transfected SW480-S/CICs and SW480-FR/CICs were compared with their NT shRNA (NT sh) transfected cells. (H–J) , * P < 0.05 was considered significant, TOPFlash/FOPFlash activity in WNT treated v6 cDNA overexpressed COS-7 cells were compared with untreated v6 cDNA transfectants; TOPFlash/FOPFlash activity in WNT plus inhibitors treated (U0126 [H], LY294002 [I], and CK 1-7 [J]) v6 cDNA overexpressed COS-7 cells were compared with inhibitors only treated v6 cDNA transfectants. (K, L) , * P < 0.05 was considered significant, TOPFlash/FOPFlash activity in v6 shRNA1 (v6 sh1) transfectant of PD-FR (CICs) (K) , and v6 cDNA overexpressed COS-7 cells (L) were compared with the NT shRNA (NT sh) transfected PD-FR (CICs) (K) , and vector control transfected COS-7 cells (L) .

Article Snippet: The M50 Super 8x TOPFlash vector (plasmid 12456) with a luciferase gene under the control of seven TCF/LEF-binding sites and the corresponding M51 Super 8x FOPFlash vector (plasmid 12457) with mutated TCF/LEF-binding sites were obtained from Addgene (Cambridge, MA, USA).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Knock-In, Clone Assay, Luciferase, Activity Assay, shRNA

Journal: Frontiers in Oncology

Article Title: Chemotherapy induces feedback up-regulation of CD44v6 in colorectal cancer initiating cells through β -catenin/MDR1 signaling to sustain chemoresistance

doi: 10.3389/fonc.2022.906260

Figure Lengend Snippet: Caveolin-mediated endocytosis is essential for CD44-LRP6- β -catenin signaling to maintain FOLFOX resistance. (A) , Detergent-resistant membranes, Triton X-100 (1%) insoluble fractions of FR and sensitive cells, were separated in the OptiPrep linear gradients, and distributions of protein and cholesterol across the gradient are shown (details in Methods). Dot Blot analyses show the presence of caviolin1 (CAV1) and clathrin in different Optiprep fractions. (B) , SW480-S and SW480-FR cells were treated with 1 x FOLFOX for 30 minutes. The raft (R) < 20% OptiPrep fractions [2-5], and the non-raft (NR) > 20% OptiPrep fractions [6-9] were isolated and analyzed by western blots (WBs) for CD44v6, phosphorylated LRP6 (S1490), LRP6, caveolin-1, and clathrin. (C) , SW480-S cells were treated with or without 5 mM methyl- β -cyclodextrin (M β CD) for 1 hour, and the R and NR fractions were analyzed by WBs for CD44v6 and clathrin. (D) , SW480-FR and SW480-S cells transfected with dominant negative dynamin (DN Dyn) [DN K44A] were co-transfected with 50 ng TOPFlash and 50 ng TK-Renilla vectors, or with 50 ng FOPFlash and 50 ng TK-Renilla vectors. After stimulation with WNT3A (20 ng/ml) for 12 hours, cells were lysed and subjected to luciferase measurements. (E) , SW480-S and SW480-FR cells were transfected with TOPFlash and TK-Renilla vectors, or with FOPFlash and TK-Renilla vectors luciferase reporter constructs. Transfected cells were treated for 1 hour with the indicated concentrations of nystatin, known to block caveolin-1-mediated endocytosis, or with monodansylcadaverine (MDC), known to block clathrin-mediated endocytosis. After stimulation with WNT3A (20 ng/ml) for 12 hour, cells were lysed and subjected to luciferase measurements. (F) , SW480-FR cells and SW480-S cells (G) SW480-S cells were treated for 1-4 hours with Nystatin (150 µg/ml) or MDC (150 µg/ml). After stimulation with WNT3A (20 ng/ml) for 1 or 4 hour, cells were lysed and subjected to western blots. Data are presented as Mean ± SD from n = 3-6 replicates in four independent experiments. All WBs data are representative of 4 independent experiments. (D) , * P < 0.05 was considered significant, TOPFlash/FOPFlash activity in DN Dyn transfectants of SW489-S, and SW480-FR cells were compared with their vector control transfectants. (E) , * P < 0.05 was considered significant, Nystatin and MDC treated SW489-S, and SW480-FR cells were compared with their untreated control cells.

Article Snippet: The M50 Super 8x TOPFlash vector (plasmid 12456) with a luciferase gene under the control of seven TCF/LEF-binding sites and the corresponding M51 Super 8x FOPFlash vector (plasmid 12457) with mutated TCF/LEF-binding sites were obtained from Addgene (Cambridge, MA, USA).

Techniques: Dot Blot, Isolation, Western Blot, Transfection, Dominant Negative Mutation, Luciferase, Construct, Blocking Assay, Activity Assay, Plasmid Preparation, Control