Article Figures & Data
Figures
- Figure 1.
Stem-like CD44+/CD24− cell population are increased in KD cells. A, expression levels of CD24 and CD44 mRNA were analyzed by qRT-PCR. After normalizing with 18S rRNA in each sample, the relative mRNA levels were calculated using control (= 1). *, P < 0.01. B, cells were costained with APC-conjugated CD24 and PE-conjugated CD44 and analyzed by flow cytometry. APC-IgG and PE-IgG were used as the negative control for gating and the labels indicated the percentage of each cell population. C, CD44 and DAB2IP mRNA (left) and protein (right) expressions were compared in CD44+ and CD44− population sorted from PZ-HPV7T cells. D, representative IHC staining of DAB2IP and CD44 in clinical specimens (left). The scale bar represents 100 μm. Right: DAB2IP and CD44 staining score in primary prostate cancer were compared with the metastatic tissue.
- Figure 2.
C2 domain of DAB2IP is the key domain for suppressing CD44 expression. A, RWPE-1 and PZ-HPV7 KD cells were cotransfected with various DAB2IP domains and CD44-luc plasmid, and then luciferase activity was determined. Asterisk indicates statistical significance in cells transfected with vector versus F, N, and PHC2 domain (P < 0.01). B, Wnt pathway–related genes expressions were compared in RWPE-1 and PZ-HPV7 Con and KD subline (left), and PZ-HPV7 WT and T, or CD44+ and CD44− population sorted from Du145 and PZ-HPV7T cells (right), respectively. C, cells were transfected with TOP for 48 hours and subjected to dual luciferase assay. D, RWPE-1 KD or PZ-HPV7 KD cells were transfected with CD44-luc plasmid for 24 hours and treated with 200 nmol/L LGK974 (left) or 5 μmol/L IWP-2 (right) for another 24 hours, then subjected to dual luciferase assay.
- Figure 3.
Characterization of the binding of β-catenin to CD44 promoter region. A, β-catenin binding to the CD44 promoter region was evaluated by ChIP assay. Chromatin DNAs prepared from PZ-HPV7 Con and KD cells were immunoprecipitated with β-catenin antibody and subjected to PCR. (
) in CD44 promoter regions. B, β-catenin plasmid was transfected to 293 cells for 48 hours and direct binding of β-catenin to the CD44 promoter was evaluated by ChIP. Rabbit IgG (immunoglobulin G) was used as the negative control. C, DAB2IP and β-catenin plasmid were cotransfected to 293 cell and ChIP assay was performed after 48 hours. D, Con cells from RWPE-1 or PZ-HPV7 were transfected with CA β-catenin mutant (S37A) for 48 hours and the expression levels of CD44 were compared using flow cytometry. - Figure 4.
CD44 is critical for PCSC development and its chemoresistance. A, characterization of shCD44 sublines of PC-3 and 22Rv1 cells generated using shRNA (Origene, TG314080) transfection by qRT-PCR and Western blot analyses. B, clonogenic assay of shCD44 or shvec cells were seeded in six-well plates at a density of 500 cells per well and cultured for 10 days then stained with crystal violet. The relative number of colony was determined by measuring OD560 nm. C, prostaspheres assay was performed for 2 weeks and the numbers of prostaspheres were compared in shvec and shCD44 cells. D, cells were seeded in a 96-well and treated with docetaxel for 48 hours. Cell viability was assessed by MTT assay.
- Figure 5.
Wnt inhibitor reduces chemoresistance of KD cells to docetaxel. A, cells were treated with docetaxel or LGK974 for 48 hours and subjected to MTT assay. B, cells were treated with 1 nmol/L docetaxel, 100 nmol/L LGK974, or combination; and cell viability was determined 48 hours after treatment by MTT assay and drug synergistic effects were determined based on combination index (CI). CI < 1, synergistic; CI = 1, additive; CI > 1, antagonistic effect. NT, nontreatment; DCT, docetaxel; LGK, LGK974; D+I, docetaxel and LGK974 combination treatment. C, the expression levels of CD44 mRNA were analyzed 48 hours after treatment by qRT-PCR. D, the prostasphere formation was determined from cells after 24-hour treatment then plated into sphere culture condition for 2 weeks. Media containing each drug were changed every 3 days.
- Figure 6.
Combination therapy targeting CD44+ population sensitizes Du145 xenograft to conventional therapy. A, Du145 cells were subcutaneously injected into SCID mice and treatment started when tumors became palpable (>100 mm3). The mice were treated with docetaxel (5 mg/kg/i.p.), LGK974 (2 mg/kg/oral gavage), or combination twice a week for 3 weeks. B, after 3 weeks treatment, xenograft tumors were excised and photographed. C, the expression levels of CD44 and β-catenin in xenograft tumors were analyzed by Western blot analysis. D, representative IHC results of CD44 in xenograft tumors after treatment were displayed. Scale bar, 100 μm.
Additional Files
Supplementary Data
- Supplemental Tables, Figures and Legends - Table S1. Primers for quantitative RT-PCR. Table S2. The list of primers for ChIP detection; CD44 promoter region. Figure S1. Loss of DAB2IP increased clonogenicity and stemness in normal prostate epithelial cells. Figure S2. Loss of DAB2IP increased in vitro invasion and motility in normal prostate epithelial cells. Figure S3. DAB2IP inhibits CD44 expression in human prostate epithelial cell lines. Figure S4. Wnt pathway correlates with CRPC progression. Figure S5. AR inhibitor doesn't affect the expression of CD44. Figure S6. All CD44 variants respond to Wnt inhibitor. Figure S7. Wnt signal regulates CD44 expression and stem cell properties. Figure S8. Wnt signal pathway mediates the expression of CD44. Figure S9. Wnt inhibitors diminish the chemo-resistance of KD cells. Figure S10. Overexpression of CD44 rescues the growth inhibition by LGK974. Figure S11. Synergistic effect of Wnt inhibitors combined with docetaxel on Du145 cells.
Volume 22, Issue 3
