Journal: bioRxiv

Article Title: A Novel miR-4745 -KLC2 Axis Regulates Cancer Stem Cell Traits in Colorectal Cancer

doi: 10.64898/2026.01.07.697660

Figure Lengend Snippet: (a) Quantification of the CD44v6-APC-positive population in CPP1 (n=7) and CTC44 (n=5) cells transfected with miR-4745-3p ( miR4745 ) or miRVana microRNA Mimic Negative Control #1 (miCTRL) mimics (right panel). Percentage of CD44v6-APC positive CPP1 cells is indicated in inset boxes for a representative experiment (left panel). (b) Representative images of tumorspheres (upper panel) and the percentage (lower panel) of sphere-forming cells in patient-derived CPP1 and circulating tumor (CTC44) colon cancer cells transfected with miR-4745-3p ( miR4745 ) or miCTRL mimics. Data from individual replicates (n=15 per experiment) are presented, along with the mean ± SEM from six independent experiments. (c) Percentage of sphere-forming cells in patient-derived CPP1 colon cancer cells transfected with miR4745 or negative control (miCTRL) mimics and, maintained as first-generation colonospheres (S1) or after several passages (S2, S3). Data from individual replicates (n=12 per experiment) are presented, along with the mean ± SEM from three independent experiments. (d) Percentage of surviving cancer stem cells (Aldefluor-positive) measured 48 hours following treatment with specified concentrations of FIRI (1X = 50 µM 5-FU + 500 nM SN38) in vitro . Sorted CPP1 Aldefluor-positive cells were first transfected with 50 nM miR-4745-3p ( miR-4745 ) or miRVana microRNA Mimic Negative Control #1 (miCTRL) mimics, 24 hours prior to FIRI treatment. Data are expressed as mean ± SEM (n = 3). (e) Percentage of Aldefluor-positive cells (% ALDH + cells) in CPP1 cells transfected with miR-4745 or miCTRL mimics and then exposed for 72 hours to chemotherapy (Firi=5µM 5-FU + 50nM SN38). Data are expressed as mean ± SEM (n=6). *, p<0.05; **, p<0.005, ***, p < 0.001, Mann Whitney test.

Article Snippet: CD44v6-APC (clone REA706, 130-111-238; Macs Miltenyi) antibody was incubated with 100 000 cells in PBS containing 5% FBS for 20 min at 4°C.

Techniques: Transfection, Negative Control, Derivative Assay, In Vitro, MANN-WHITNEY

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: FOLFOX induces CD44v6 expression. (A) , QPCR data for CD44v6 expression in 6 CRC cell lines are shown as fold change relative to pre-neoplastic Apc 10.1 cells as controls. (B–E) , The concentrations (IC 50 ) (µg/ml) of (5-FU) and (OXA) required to achieve a 50% growth inhibition relative to untreated controls using the ATP Glo-growth assay are shown (B) . The IC 50 values of 5-FU (C) , OXA (D) , and FR (E) for sensitive (S) and corresponding FOLFOX resistant (FR) cells are shown. (F) , Real-time PCR data for the CD44v6 expression are presented for SW480 tumor cells resistant to either 5-Fluorouracil (5FUR), oxaliplatin (OXAR), or FOLFOX (FR) compared to sensitive (S) pairs of cells. Gene expression was normalized to the reference gene GAPDH. (G) , Constant and variable exons are shown for the PCR primers used to amplify CD44 variable (v) and standard (s) isoforms in the human CD44v6 gene. The primers for both the v6 and standard isoforms (CD44s) predominantly generate one PCR product c5v6c7 (v6) for CD44v6 and C5C7 for CD44s, whereas the primers for the v8 variants amplify two splice variants C5v6v7v8C7 (v6-v8) and C5v8C7 (v8). These PCR products are depicted in panel H and panel I experiments. (H) , RT-PCR results are shown for the CD44v isoforms using the different primers in the tumor cells derived from colorectal patients (PD) who were resistant to 5FU, OXA and FOLFOX (PD-5-FU, PD-OXA and PD-FR). (I) , RT-PCR results are shown for the CD44v isoforms using the different primers (C5v6v8v9C7, C5v6v8C7 and C5v6C7) in the PD-FR, PD-OXA and PD-5-FU cells. Data are presented as Mean ± SD from n = 3-6 independent replicates in three independent experiments. All semi quantitative RT-PCR data are representative of three experiments (C–E) , *P < 0.05 was considered significant, 50% inhibitory concentration (IC 50 ) for 5-FU in SW480-5FUR cells was compared with SW480-S cells (C) , for OXA in SW480-5FUR cells was compared with SW480-S cells (D) , and for FOLFOX n SW480-5FUR was compared with SW480-S cells (E, F) , * P < 0.05 considered significant, fold expressions of CD44v6mRNA in SW480-5FUR, SW480-OXAR, and SW480-FR cell were compared to control (SW480-S) cells.

Article Snippet: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

Techniques: Expressing, Inhibition, Growth Assay, Real-time Polymerase Chain Reaction, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Quantitative RT-PCR, Concentration Assay, Control

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: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

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: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

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: CD44 exon specific PCR examined using 5’ primers complementary to individual variable exons and a primer to the 3’ constitutive exon 7.

Article Snippet: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

Techniques: Sequencing

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: CD44 exon specific PCR examined using 3’ primers complementary to v6 and v8 exons and a primer to the 5’ constitutive exon 5.

Article Snippet: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

Techniques: Sequencing

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: Real-time PCR (QPCR) primers for various genes associated with CICs stemness function.

Article Snippet: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

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: shRNA sequence in pSico and pSicoR vectors ( https://web.mit.edu/jacks-lab/protocolsl ).

Article Snippet: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

Techniques: shRNA, Sequencing, Luciferase

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: Cis-sequences bourd by CD44v6.

Article Snippet: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

Techniques: Clone 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: Nuclear CD44v6 associates with TCF4 and functions to modulate MDR1 transcription in FOLFOX resistant cells. (A) , The intracellular domain (ICD) of CD44v6 induces 14 C Oxaliplatin Efflux/Retention in SW480-S and SW480-FR cells by FOLFOX and WNT3A treatments. For analyzing drug retention, tumor cells were transfected for 48 hours with CD44v6shRNA, or with a CD44 Δ67 construct (devoid of the ICD region of CD44v6). They were then treated with 14 C-oxaliplatin for 24 hours, washed and incubated in drug-free medium alone or with 1 x FOLFOX, or with WNT3A (50 ng/ml) for 12 hours. Cells were harvested and washed, and their numbers were measured by a coulter counter. The radioactivity associated with cells (indicated as intracellular drug retention) were then measured by a liquid scintillation counter as described in Methods. (B) , QPCR analyses of CD44v6, β -catenin and MDR1 levels in SW480-S, SW480-FR, SW480-OXAR and SW480-5FUR cells are shown. (C) , QPCR analyses of CD44v6 and MDR1 mRNAs in SW480-S, SW480-5FUR and SW480-OXAR cells treated with or without 1 x FOLFOX or 20 ng/ml WNT3A for 12 hours are shown. (D-E) , QPCR analyses are shown for TCF4 , CD44v6 or MDR1 levels in SW480-S cells overexpressing constitutively active (CA) pTCF4 cDNA (D) , or for β -catenin, CD44v6 or MDR1 levels in SW480-S cells overexpressing pCA- β -catenin (E) . (F-G) , Transcription activities of the MDR1 promoter with TCF4 binding sites were measured using the indicated pGL3 reporters. (F) , The scheme shows the constructs with TCF binding sites in the pGL3 MDR1 promoter. (G) , MDR1 Luciferase activity reporter assays are shown for SW480-FR cells overexpressing shRNA for NT (Control), or β -catenin, or CD44v6, or a dominant negative pTCF4-DN construct. (H-I) , MDR1 gene expressions regulated by TCF4 in SW480-FR cells are shown. (H) , The sketch map shows the predicted TCF4 binding sites (CTTTGA) within the indicated MDR1 promoter. The transcriptional start site was at +1, and ATG is at the translation start site. The putative TCF4 binding sites ( MDR1 [A], MDR1 [B] and MDR 1 [C]) are shown, and their locations are labeled. (I) , Semiquantitative PCR products using ChIP PCR primers for MDR1 (A–C) were amplified. (J) , ChIP assays were done using anti-CD44v6 (red), anti-TCF4 (purple), anti- β -catenin (blue), or irrelevant IgG antibody (green) as negative control using indicated ChIP primers in SW480-FR cells. Total genomic DNA was used as control for the PCR. Quantitative qPCR data representing the qPCR products in immunoprecipitated DNA versus 10% input DNA of ChIP primers for the designated TCF4 binding sites on MDR1 [A], MDR1 [B] and MDR 1 [C] are shown. (K) , ChIP assays were done using either anti-CD44v6 (red), anti-TCF4 (purple), anti β -catenin (blue), or irrelevant IgG antibody (green) in SW480-FR cells overexpressing CD44v6 shRNA1, or NT-shRNA1, or with pCD44v6 WT, or pCD44v6 NLS mutant constructs. Quantitative ChIP-QPCR data representing the PCR products in immunoprecipitated DNA versus 10% input DNA of ChIP primers for the designated TCF4 binding sites on MDR1 (A, H) are shown. QPCR and ChIP PCR data represent mean +/- SD, n = 5 replicates from at least 3 independent experiments. (B) , * P < 0.05 was considered significant, CD44v6, β -catenin, and MDR1 mRNA levels in 5-FUR, OXAR, and FR SW480 cells were compared with SW480 sensitive cells. (C) , * P < 0.05 was considered significant, CD44v6, and MDR1 mRNA levels of FOLFOX and WNT treated cells were compared with vehicle controls in each cell type. (D) , * P < 0.05 was considered significant, CD44v6, TCF4 and MDR1 mRNA levels of CA-TCF4 transfectant were compared with vector transfectant. (E) , * P < 0.05 was considered significant, CD44v6, β -catenin, and MDR1 mRNA levels of CA- β -catenin transfectant were compared with vector transfectant. (G) , * P < 0.05 was considered significant, CD44v6, β -catenin, and MDR1 mRNA levels of CA- β -catenin transfectant were compared with vector transfectant. (G) , Luciferase data in “ G” represent results from 3 independent experiments performed in triplicates. * P < 0.05 was considered significant, Luciferase activity of TCF4 DN. β -catenin sh1, and CD44v6 sh1 transfectant of SW480-FR cells for all the PGL3-mdr1 (A) , PGL3-mdr1 (B) , and PGL3-mdr1 (C) , constructs were compared with that of vector control. (J) , * P < 0.05 was considered significant, ChIP PCR data for all MDR1 sites (A–C) of TCF4. β -catenin, and CD44v6 antibody data were compared with that of IgG control in SW480-FR cells. (K) , * P < 0.05 was considered significant, ChIP PCR data for MDR1 (A) site of TCF4. β -catenin, and CD44v6 antibody data were compared with that of IgG control in v6 shRNA1 (v6 sh1) and v6 NLS Mut transfectant of SW480-FR cells were compared with respective controls such as NT shRNA, and v6 cDNA transfectant of cells.

Article Snippet: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

Techniques: Transfection, Construct, Incubation, Radioactivity, Binding Assay, Luciferase, Activity Assay, shRNA, Control, Dominant Negative Mutation, Labeling, Amplification, Negative Control, Immunoprecipitation, Mutagenesis, ChIP-qPCR, 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: Nuclear TCF4 modulates CD44v6 transcription in resistant cells, and the ICD domain of CD44v6 is required for oxaliplatin (a component of FOLFOX) efflux. (A-D) , CD44v6 was transcriptionally regulated by TCF4 in SW480-FR cells. (A) , The sketch maps of predicted TCF4 binding sites (CTTTGA) within the CD44v6 luciferase promoter (A) , and CD44v6 (B) are shown in SW480-FR CICs. (B) , CD44v6 luciferase (Luc) activity reporter assays are shown for SW480-FR cells CICs overexpressing dominant negative (DN) TCF4, or β -catenin shRNA, or NT-shRNA (control). (C, D) . (C) The sketch map shows the predicted TCF4 binding sites (CTTTGA) within the indicated CD44 promoter. The transcriptional start site was at +1, and ATG is at the translation start site. The putative TCF4 binding sites (CD44v6 [A], CD44v6 [B]) are shown, and their locations are labeled by blue arrows. (D) Semiquantitative PCR products using ChIP PCR primers for the designated TCF4 binding sites on CD44v6 (A) and CD44v6 (B) , were amplified in SW480-FR CICs. (E) , ChIP-qPCR using PCR primers for designated CD44v6 (A) sites (as shown in the schematic diagram in (C) ) were used for amplification of the CD44v6 mRNA of untreated SW480-FR CICs cells and of CICs overexpressing the indicated vector and TCF4-DN cDNA. (F) Proposed model is shown for a positive feedback loop coupling β -catenin/TCF4 activation and CD44v6 alternate splicing that sustains cancer initiating cell proliferation and FOLFOX resistance. Left panel: In FOLFOX-resistant cells, in the absence pf DAB2, elevated WNT3A induces CD44v6 that recruits LRP6 to caveolin-micro domain through its nuclear localization site (NLS). The CD44v6-lRP6 complex is internalized through the caveolin-mediated endocytosis followed by endosomal sorting, resulting in accumulation of a TCF4-CD44v6 complex that causes transcriptional activation and the expression of its target genes including CD44v6 and MDR1 genes. Our results are the first demonstration of a positive feedback loop linking FOLFOX mediated increased WNT3A signaling-dependent alternative splicing of CD44 which is important for cell cycle progression resulting in FOLFOX-resistance in CRC-CICs in the absence of DAB2. Right panel: In sensitive cells in the presence of DAB2, the CD44v6-LRP6 complex is internalized through the clathrin-mediated endocytosis pathway and promotes β -catenin destruction and fails to recruit the β -catenin/TCF4-CD44v6 complex into the nucleus. Data in B, E represent at least 3 independent experiments performed in triplicates. Values represent means ± SD.; n = 3–5; Semi-quantitative PCR data in “D” are representative of three experiments. (B) , * P < 0.05 was considered significant, Luciferase activity of TCF4 DN. and β -catenin sh1 transfectant of SW480-FR cells for all the PGL3-CD44v6 (A) , and PGL3-CD44v6 (B) constructs were compared with that of vector control. (E) , * P < 0.05 was considered significant, ChIP PCR data for CD44v6 (A) sites of TCF4. and β -catenin antibody data were compared with that of IgG control in SW480-FR cells.

Article Snippet: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

Techniques: Binding Assay, Luciferase, Activity Assay, Dominant Negative Mutation, shRNA, Control, Labeling, Amplification, ChIP-qPCR, Plasmid Preparation, Activation Assay, Expressing, Alternative Splicing, Real-time Polymerase Chain Reaction, Transfection, Construct

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: ChiP PCR primers for MDR1and CD44v6 promoters.

Article Snippet: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

Techniques: Sequencing

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: Tumor response to FOLFOX is correlated with enrichment of CD44v6 (+) CICs and resistance of CICs. (A) , Percentages of tumor sphere formation of PD-FR CICs and Non-CICs were measured in a sphere-formation assay. Representative pictures of tumors are shown. (B) , Implantation of 5 x 10 3 CICs from SW480-FR (SQ) tumors resuspended in Matrigel were tumorigenic while 100-fold more unsorted cells (Bulk) resuspended in Matrigel were required to generate tumors in four independent implantations. Five mice per group were used. SW480-FR SQ tumor weights following injection of indicated cell numbers from CICs and unsorted (Bulk) tumor cells are shown. (C) , A representative image of tumors initiated from (B) is shown. (D) , FACS sorted 2 x 10 3 CICs resuspended in Matrigel were tumorigenic while 250-fold more unsorted bulk cells (resuspended in Matrigel) were unable to generate the same capacity of tumorigenesis in four independent specimens. Six mice per group were used for four independent experiments. (E) , Numbers of SQ tumors formed by implantations of the indicated numbers of CICs and Non-CICs resuspended in Matrigel that were FACS sorted from the SW480-FR cells and PD-FR cells are shown. Five mice were used per group. (F) , FACS sorted CICs (2 x 10 3 ), Non-CICs (5 x 10 5 ), and the unfractionated bulk tumor cells (5 x 10 5 ), from indicated tumor samples were resuspended in Matrigel and implanted in immunocompromised mice. The same cells from the first generation of SQ tumors were further implanted into immunocompromised mice. The experiments were repeated to generate tumors into the third generation of xenograft tumors. Only CICs and the unfractionated bulk tumor cells were capable of inducing tumor formations. Isolation of second and third generation xenograft tumor cells displayed similar results (n = 5 mice; experiments were performed in triplicates). (G) , Tumor volumes from the experiment (F) were measured in implanted tumors from the indicated CICs, and from the unfractionated bulk tumor cells (n = 5 mice; experiments were performed in triplicates). (H) , Percentages of tumor sphere formation in freshly isolated dissociated primary, secondary and tertiary xenograft tumors generated with the indicated CICs, Non-CICs, and unsorted bulk tumor cells from the experiment (F) are shown. (I) , Enrichments of CICs in bulk cells from three sources – 1) patient derived specimens, 2) SQ tumor samples developed using FR-cells, and 3) the corresponding sensitive pairs, were assessed by FACS analysis for CD44v6 after FOLFOX treatment. Data are representative of four independent human specimens, and of three independent tumor samples from sensitive and FR cells. Data are presented as Mean ± SD from n = 3 replicates in three independent experiments. (A) *P < 0.05, were considered significant, tumor sphere growth in PD-FR CICs were compared with Non-CICs. (B) , *P < 0.05, **P < 0.01, were considered significant, SQ tumor growth in SW480-FR CICs were compared with bulk tumor from SW480-FR cells. Tumor growth kinetics data in (B, C) , n = 6 mice/each group represent mean +/- SD, from at least 3 independent experiments. (D, E) , n = 6 mice/each cell types (CIC or unsorted) in each cell types in three independent experiments represent mean +/- SD. (F, G) , n = 5 mice/each cell types (CIC, Non-CICs, or unsorted) in three independent experiments represent mean +/- SD, *P < 0.05, **P < 0.01, were considered significant, tumor growth using CICs were compared with tumors from bulk tumor cells. (H) , *P < 0.05, *P < 0.05, *P < 0.05, were considered significant for tumor sphere growth from cells from primary, secondary, and tertiary xenografts of CICs compared with bulk cells. Tumor growth kinetics data in L (n = 7) represent mean +/- SD, from at least 3 independent experiments.

Article Snippet: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

Techniques: Tube Formation Assay, Injection, Isolation, Generated, Derivative 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: FOLFOX induced CD44v6 expression establishes FOLFOX (FR) resistant colorectal cancer cells (CRCs). (A) , Schematic illustration of the CD44 gene. Both constitutive (c) and variable (v) exons are represented. The PCR primers used to amplify CD44 variable and standard isoforms are shown as arrows and the PCR products are depicted in panel (B) experiments. (B) , Serum-starved SW480-S cells were stimulated with 1 x FOLFOX (50 µg/ml 5-flurouracil + 10 µM oxaliplatin + 1 µM leucovorin) at the indicated time periods. The mRNA expressions show that primers for v6 isoforms generate v6 and v6-v8 PCR products. The primers for v8 and the standard isoform of CD44 primarily generate one product. (C) , Western blots are shown for antibodies that recognize either CD44v6, the active hypo phosphorylated β -catenin (Active β -catenin (ABC)), β -catenin, MDR1 or β -tubulin in sensitive (S) and FR clones of SW480 cells following stimulation with 1x FOLFOX for 4-48 hours. (D, E) , Upper panels: Semi-quantitative RT-PCR analyses are shown for CD44 variants in SW480-S and SW480-FR cells transfected with pSicoR-non targeted shRNA1 (NT sh1) or pSicoR-v6 shRNA1 (v6 sh1) (D) , and NT sh2 or v6 sh2 (E) for 72 hours followed by FOLFOX stimulation for another 12 hours. Lower Panels: Western blot analyses are shown for p-LRP6 (S1490), active ABC, β -catenin, MDR1, or β -tubulin following 1 x FOLFOX stimulation for 12 hours in cells transfected with NT sh1 or v6 sh1. (F) , Effects of CD44v6 shRNA1 and WNT3A shRNA1 knockdown of CD44v6 and WNT3A respectively on the viability of SW480-FR cells in presence of v6 cDNA that were treated with various concentrations of FOLFOX. An ATP based assay (CellTiter-Glo) measured cell viability compared with vector transfectant without FOLFOX treatment as 100%. Error bars represent calculated SDs (n = 3). (G) Same experiments as in F carried out in presence of CD44v6 shRNA2 and WNT3A shRNA2. (H) Sixteen hours after release from G2/M phase by nocodazole treatment, when 97% of the cells were in G1 phase (cell cycle analyzed by flow cytometry) the cells were transfected with either non-targeted (Control) or CD44v6 shRNA. Twenty four hours after transfection, the cells were incubated in low serum medium (0.5% serum) with 1 x FOLFOX for different time periods to re-enter the cell cycle. Cell cycle analysis were carried out in these cells. Samples were analyzed through a time course of 12 hours. Bar graph summarizing the flow cytometry cell cycle profile analysis of SW480 cells. Error bars represent ± SEM of five independent experiments. (I) Upper and lower panels: Western blot for the activated β -catenin accumulation in nuclear fraction in G2/M arrested and 1 x FOLFOX or 20 ng/ml WNT3A stimulated SW480-cells which were previously transfected with NT shRNA or v6 shRNA for 24 hours. These cells were collected at indicated times after 1 x FOLFOX or WNT3A -stimulation. Inset: A model for sustained WNT3A signaling and G1–S transition, dependent on CD44 v6 variants (details are in the text). (J) , Anchorage-independent growth in soft agar is shown for SW480-FR, WIDR-FR and LOVO-FR cells and compared with their sensitive (S) pairs. Scale bars, 100 μm. (K) , Tumor-sphere formation assays were done for the SW480-FR, WIDR-FR and LOVO-FR cells and compared with their sensitive (S) pairs. Scale bars, 100 μm. (L) , Tumor formation is shown in nude mice injected with either 5 x 10 4 SW480-FR cells, or 5 x 10 4 SW480-S cells, or 1 x 10 6 SW480-S cells. SW480-FR cells formed tumor nodules in all injected mice (7/7). Starting at week 3, SW480-R (5 x 10 4 ) cells induce tumor nodules whereas SW480-S (5 x 10 6 ) cells induced much smaller tumor nodules starting a week later than the SW480-FR cells (7/7 mice). SW480-S (5 x 10 4 ) cells were unable to induce tumors. Growth curves are shown for these xenograft tumors in immunocompromised mice. Data are presented as Mean ± SD from n = 3-6 replicates in three independent experiments. All WBs, FACS data, semi quantitative RT-PCR data are representative of three experiments (F, G) , *P < 0.05, **P < 0.01 were considered significant, percent cell viability in WNT3A shRNA and CD44v6 shRNA transfected cells compared with vector control and NT shRNA transfected. *P < 0.05, **P < 0.01 were considered significant, percent cell viability in WNT3A shRNA + v6 cDNA transfected cells compared with WNT3 shRNA transfected cell. Student’s t-test was used to assess the significance. (H) , *P < 0.05, **P < 0.01 were considered significant, percent cells in S phase in CD44v6 shRNA transfected cells compared with NT shRNA transfected cell. (J–L) *P < 0.05, **P < 0.01, ***P < 0.001 were considered significant. Soft agar colonies, tumor sphere growth, and SQ tumor growth of SW480-FR cells were compared with SW480-S cells. Tumor growth kinetics data in (L) (n = 7) represent mean +/- SD, from at least 3 independent experiments.

Article Snippet: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

Techniques: Expressing, Western Blot, Clone Assay, Quantitative RT-PCR, Transfection, Knockdown, ATP Assay, Plasmid Preparation, Flow Cytometry, Control, shRNA, Incubation, Cell Cycle Assay, Injection

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 identifies colorectal cancer (CRC) initiating cells (CICs). (A) , Single-cell suspensions from patient derived (PD) specimens collected from 5-FU (PD-5FUR), Oxaliplatin (PD-OXAR) and FOLFOX (PD-FR) resistant tumor tissues, and from SW480-FR/subcutaneously (SQ) tumor cells were processed for tumor spheres. Sphere propagated cells were FACS sorted for high expressions of CD44v6-PE. CD44v6 positive (+) populations were sorted using EpCAM (–) FITC and CD44v6-PE. CD44v6 (+)/EpCAM (+) cells from various tumors were cultured separately and grown in fresh medium for 2 weeks. The cells were then subjected to flow cytometric analysis for isolation of CD44v6 (+)/EpCAM (+)/ALDH1 (+)/CD133 (+) (CICs), and for CD44v6 (–)/EpCAM (+)/ALDH1 (+)/CD133 (+) (Non-CICs) using antibodies to ALDH11-FITC, CD133-FITC or CD44v6-PE. (B–D) , Percentages of CD44v6 (+) and CD44v6 (–) fractions in EpCAM (+) (B) , in EpCAM (+)/ALDH1(+) (C) , and in EpCAM (+)/ALDH1 (+)/CD133 (+) (D) sorted cells in PD-FR tumor tissues are shown. Henceforth, the CD44v6 (+)/EpCAM (+)/ALDH1 (+)/CD133 (+) cells are identified as CICs, and the CD44v6 (–)/EpCAM (+)/ALDH1 (+)/CD133 (+) cells as Non-CICs (details in Methods). (E) , QPCR analyses of CIC-stemness markers (ALDH1, SOX2, OCT4, Nanog, c-Myc and CD44v6) were done on PD-FR CICs, PD-FR Non-CICs, SW480-FR CICs, and SW480-FR Non-CICs isolated from SW480-FR and SW480-S SQ tumor samples. (F) , CD44v6 mRNA expressions (by QPCR) are shown in PD-FR, PD-OXAR and PD-5FUR cells treated with or without 1 x FOLFOX for 12 hours. Data are presented as fold change of CD44v6 mRNA expressions relative to adjacent control cells from colon tissue. (G, H) , Cell viability of CICs and non-CICs from three independent PD-FR (G) and PD-5-FUR (H) cultures following treatment with or without FOLFOX were assessed by an ATP based assay (CellTiter-Glo). (I–J) , Apoptosis of CICs and non-CICs from three independent PD-FR (I) and PD-5-FUR (J) cultures following 1 x FOLFOX treatment were assessed by a Caspase 3 ELISA assay. Data are presented as Mean ± SD from n = 3-6 replicates in three independent experiments. All QPCR, and FACS data are representative of three independent experiments (B–D) , *P < 0.05, were considered significant, CD44v6 (+) cells were compared with CD44v6 (–) cells. (E) , *P < 0.05, were considered significant, expression of stemness associated factors of CICs were compared with Non-CICs. (FH–J), *P < 0.05, were considered significant, FOLFOX treated cells were compared with Non FOLFOX cells.

Article Snippet: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

Techniques: Derivative Assay, Cell Culture, Isolation, Control, ATP Assay, Enzyme-linked Immunosorbent Assay, Expressing

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: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

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: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

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: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

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

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: DAB2 favorably sequesters a CD44v6-LRP6 complex in the direction of clathrin-dependent endocytosis to retain FOLFOX sensitivity. (A) , mRNA expressions of disheveled protein 2 (DVL-2) and DAB2 protein normalized to β -actin in sensitive and resistant pairs of cells are shown by qPCR. (B) , Whole cell lysates (WCL) of Vector and DAB2 cDNA transfected SW480-FR cells at time 0 or after 12 hours treatment with or without WNT3A were analyzed by WB for the indicated proteins. Vector (C) , and DAB2 cDNA (D) transfected SW480-FR cells were treated with or without WNT3A for 12 hours, and the Triton X-100 (1%) insoluble fractions were separated into lipid raft (R) and non-raft (NR) by OptiPrep gradient centrifugation. Pooled OptiPrep gradient fractions (light fractions (3–4) and non-raft heavy fractions (7–8)) were immunoprecipitated with anti-caveolin-1 (left panels) and anti-clathrin (right panels) antibodies. Immunocomplexes were immunoblotted for CD44v6, LRP6 (S1490), LRP6, Axin, GSK3 β , and DAB2, and for clathrin or caveolin (bottom panels). IgG antiserum was used as negative control for the various immunoprecipitations. QPCR data represent results from 3 independent experiments done in n = 3-6 replicates. All WB data are representative of 4 independent experiments. (A) , * P < 0.05 was considered significant, DVL2 and Dab2 mRNA expressions in FR cells were compared with S cells. (B) , * P < 0.05 was considered significant, DVL2 and Dab2 mRNA expressions in LGK974 treated SW480-FR cells were compared with untreated SW480-FR cells.

Article Snippet: To enrich cells for CICs, single cells were labeled with a phycoerythrin (PE)-conjugated monoclonal antibody against CD44v6 (Miltenyi Biotec), and then analyzed for the expression of Fluorescein-5-isothiocyanate (FITC) with conjugated monoclonal antibody against EpCAM (R&D Systems).

Techniques: Plasmid Preparation, Transfection, Gradient Centrifugation, Immunoprecipitation, Negative Control