CD44v8-10 is a marker for malignant traits and a potential driver of bone metastasis in a subpopulation of prostate cancer cells

Cytokeratin expression in PC3 subpopulations. (A) Representative immunofluorescence assay by using DAPI for nuclei staining (blue signal) and antibody for pan-cytokeratins (rhodamine, red signal). (B) Flow cytometry analysis using antibodies for pan-cytokeratins and (C) for CK18. Mean ± standard error of mean derived from the value of the median fluorescence of 3 independent experiments (B, right) and from the percentage of CK18-positive cells (C, right) of 3 independent experiments. **P < 0.01; ***P < 0.001, Student’s paired t-test. (B and C, left) Representative experiments of flow cytometry analysis for indicated cytokeratins.

Expression of epithelial/mesenchymal markers and reactive oxygen species (ROS) production in PC3 subpopulations. CD44v8-10^pos cells show high levels of E-cadherin (A) and EpCAM (B) and low levels of vimentin (C) and N-cadherin (D) compared with CD44v8-10^neg cells. (Middle panels) Histograms of E-cadherin (A), EpCAM (B), and vimentin (C) of 3 independent experiments, and the respective representative flow cytometry plots are shown in the left panels of A, B, and C. The levels of E-cadherin (A, right) and vimentin (C, right) mRNA assayed by real-time quantitative polymerase chain reaction analysis mirror the protein levels. (D, left) Whole-cell extracts of CD44v8-10^pos and CD44v8-10^neg PC3 cells were analyzed by Western blot for N-cadherin. β-Actin was used as control for equal amounts of proteins loaded. Each blot is representative of three experiments. (D, right) Histogram of the densitometric analysis of the Western blot of 3 separate experiments representing the fold decrease of the N-cadherin/β-actin ratio (CD44v8-10^neg value set as 1). (E) Endogenous ROS levels were measured by flow cytometry using DCFDA dye fluorescence in cells treated with or without 200 μM hydrogen peroxide (H₂O₂) for 30 min. The graph presents 3 independent experiments and a representative flow cytometry histogram. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s paired t-test.

Association between CD44v8-10 expression and the prognosis of patients with prostate cancer (PC). Adhesion ability to type I collagen of PC3 cell populations. (A) CD44v8-10 expression (ENST00000433892.6) was analyzed using GEPIA software in normal/benign samples (from TCGA and GTEx, green bar) and in prostate adenocarcinoma (PRAD from TCGA, red bar). Cancer tissue samples (n = 492) and normal tissues (n = 152). *P < 0.05, Student’s t-test. (B) Analysis of patients with PC collected by the TCGA dataset shows CD44v8-10 expression in the iClusters: iCluster1, mild prognosis; iCluster2, intermediate prognosis; iCluster3, poor prognosis. (C) The 2 PC3 subpopulations were cultivated on type I collagen-coated plates for 90 min. After staining with crystal violet, the cells were detached and the absorbance was determined using a spectrophotometer. (D) Flow cytometry analysis for α2β1-integrin, type I collagen receptor, and the relative representative flow cytometry histogram. Data represent the mean ± standard error of mean derived from 3 independent experiments. *P < 0.05; **P < 0.01; Student’s paired t-test.

Expression analysis of genes involved in osteomimicry process and functional assay for osteoblast-like activity. (A) Real-time quantitative polymerase chain reaction analysis for genes encoding proteins involved in osteoblast and osteoclast differentiation and function. β-actin was used as an internal control. (B, left) Whole-cell extracts of CD44v8-10^pos and CD44v8-10^neg PC3 cells were analyzed by Western blot for RUNX2. β-actin was used as control for equal amounts of proteins loaded. Each blot is representative of 3. (B, right) The histogram represents the densitometric analysis of Western blot of 3 separate experiments representing the fold increase of the RUNX2/β-actin ratio (CD44v8-10^neg value set as 1). (C, left) CD44v8-10^pos and CD44v8-10^neg PC3 cell ability to release mineralized nodules revealed by von Kossa staining and (D, left) to form calcium deposits revealed by Alizarin Red staining. (C and D, right) Mean ± standard error of mean derived from 3 independent experiments performed as described in the left. *P < 0.05; **P < 0.005; ***P < 0.001 vs. CD44+, Student’s paired t-test.

Effects of conditioned medium (CM) from CD44v8-10^pos and CD44v8-10^neg PC3 cells on osteoblasts and osteoclastogenesis and effect of CD44v8-10 siRNA on osteomimicry genes expressed by CD44v8-10^pos PC3 cells. Healthy-donor osteoblasts were treated 48 h with 100% CM obtained from 2 different PC3 cultures and cytochemical analysis of alkaline phosphatase (ALP) activity of osteoblasts was performed. Representative ALP staining (A, left) and densitometric analysis of ALP staining of 3 independent experiments evaluated as arbitrary units (A.U.) (A, right). Results are expressed as mean ± standard error of mean (SEM). *P < 0.05. (B) Healthy-donor peripheral blood mononuclear cells (PBMCs) were treated with 50% CM obtained from 2 different PC3 cultures and TRAcP staining was performed. In the representative images, the osteoclasts are indicated by red arrows. (C) Number of multinucleated (> 3 nuclei) TRAcP-positive osteoclasts (OC) and (D) number of nuclei per osteoclast of 3 independent experiments were evaluated. Results are expressed as mean ± SEM. *P < 0.05. (E) Three healthy-donor PBMCs were treated with 50% CM obtained from 3 different PC3 cultures treated with CD44v8-10 siRNA/scramble and TRAcP staining was performed. In the representative images the osteoclasts are indicated by red arrows. (F) Histograms show number of multinucleated (> 3 nuclei) TRAcP positive osteoclasts (OC) in the experimental setting described in panel E. Results are expressed as mean ± SEM. *P < 0.05; ***P < 0.001 vs. scramble CD44v8-10^pos-derived CM treatment. Effect of CD44v8-10 siRNA on the expression of the indicated osteomimicry genes at mRNA (G) and protein (H) levels. Each Western blot is representative of three experiments. β-actin was used as control for equal amounts of proteins loaded. The histograms show the densitometric analysis of Western blot of 3 separate experiments representing the relative expression being scramble RNA value set as 1. *P < 0.05; ***P < 0.001.

Involvement of Wnt and TAZ pathways in CD44v8-10-mediated signaling. Whole-cell extracts of CD44v8-10^pos and CD44v8-10^neg PC3 cells were analyzed by Western blot for Phospho-β-catenin and total-β-catenin (A, left). β-Actin was used as control for equal amounts of proteins loaded. Each blot is representative of 3. The histograms represent the densitometric analysis of Western blot of 3 separate experiments representing the fold decrease (CD44v8-10^neg PC3 cells vs. CD44v8-10^pos PC3 cells) of the phospho-β-catenin/β-catenin ratio (A, right). (B) Representative immunofluorescence assay by using DAPI for nuclei staining (blue signal) and antibody for β-catenin (fluorescein, green signal). (C) TAZ and its targets expression levels were evaluated by real-time quantitative polymerase chain reaction in the 2 PC3 cell populations in basal conditions and (D) in CD44v8-10^pos PC3 cells after treatment with CD44v8-10 siRNA. Results are expressed as mean ± standard error of mean. *P < 0.05; ***P < 0.001.