Centrosomal PKCbetaII and pericentrin are critical for human prostate cancer growth and angiogenesis - PubMed (original) (raw)

Centrosomal PKCbetaII and pericentrin are critical for human prostate cancer growth and angiogenesis

Jeewon Kim et al. Cancer Res. 2008.

Erratum in

Abstract

Angiogenesis is critical in the progression of prostate cancer. However, the interplay between the proliferation kinetics of tumor endothelial cells (angiogenesis) and tumor cells has not been investigated. Also, protein kinase C (PKC) regulates various aspects of tumor cell growth, but its role in prostate cancer has not been investigated in detail. Here, we found that the proliferation rates of endothelial and tumor cells oscillate asynchronously during the growth of human prostate cancer xenografts. Furthermore, our analyses suggest that PKCbetaII was activated during increased angiogenesis and that PKCbetaII plays a key role in the proliferation of endothelial cells and tumor cells in human prostate cancer; treatment with a PKCbetaII-selective inhibitor, betaIIV5-3, reduced angiogenesis and tumor cell proliferation. We also find a unique effect of PKCbetaII inhibition on normalizing pericentrin (a protein regulating cytokinesis), especially in endothelial cells as well as in tumor cells. PKCbetaII inhibition reduced the level and mislocalization of pericentrin and normalized microtubule organization in the tumor endothelial cells. Although pericentrin has been known to be up-regulated in epithelial cells of prostate cancers, its level in tumor endothelium has not been studied in detail. We found that pericentrin is up-regulated in human tumor endothelium compared with endothelium adjacent to normal glands in tissues from prostate cancer patients. Our results suggest that a PKCbetaII inhibitor such as betaIIV5-3 may be used to reduce prostate cancer growth by targeting both angiogenesis and tumor cell growth.

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Figures

Figure 1

Figure 1

PKCβII is active in growing PC-3 prostate tumors and is localized mainly in tumor endothelium as compared with other PKC isozymes. (A) The level of the active form of PKC isozymes was determined by Western blot analyses of cytosolic (C) and particulate (P) fractions from 3-, 4- and 6- week-old tumors using anti-PKCα, βI, βII and ε antibodies. Tumors were fractionated as described in Methods. Normal human prostatic epithelial cells (PEC) grown in serum-free medium (Complete PFMR-4A (24)) without bovine pituitary extract, were used to show basal levels of PKC translocation in this cell type. Quantification of the active forms of PKCβI and βII at week 6 (translocation; expressed as percentage of PKC isozyme in the particulate fraction over total cellular enzyme) is provided on the right (n=4, *; p=0.01). A 2-tailed Student’s t test was used to determine significance. Loading controls for cytosolic and particulate fractions (GAPDH and Gαi) are shown. (B) Immunofluorescence staining of PC-3 prostate tumors 6 weeks after tumor implantation in mice demonstrated different levels of PKC isozymes in tumor vessels. Representative immunostaining using anti-PKCα, βI, βII, ε antibodies (red, top), anti-CD31 antibodies (green, middle) and merged images (yellow, bottom, arrow heads) are shown (n=5 each). Scale bar 10 μm.

Figure 2

Figure 2

In the early phase of tumor growth, an increase in endothelial cell proliferation rate precedes that of the tumor cells. (A) PC-3 tumor cells (5×106 cells) were injected s.c. into the left flank and the xenograft tumors were isolated each week up to 6 weeks after tumor implantation. Deuterated water was administered via i.p. injection (8%) and in the drinking water (4%) for 1 week prior to each study. (B) Tumor volume of PC-3 xenografts from week 1 to week 6 after tumor cell injection was measured using a caliper (mean±SEM). (C) Proliferation rates of isolated tumor endothelial cells (open circle) and tumor cells (filled circle) were analysed by GC-MS (n=4–7 per week). Different cell populations were isolated using FACS (see supplemental Figure 1). Proliferation rate [i.e. fractional turnover rate (k) per day] was calculated as previously described (28, 29). Repeated ANOVA was used to determine the significance of differences between the curves. A 2-tailed Student’s t test and ANOVA were used to determine the differences (p<0.005, repeated ANOVA; #; p<0.05, Student’s t test). (Insert) The xenograft tumors were grown for 4 and 7 days after tumor cell injection and tumor endothelial cells and tumor cells were obtained to measure their proliferation rates. Deuterated water was administered for 4 days before sacrifice (n=6–10 per time point).

Figure 3

Figure 3

PC-3 tumor growth rate was reduced with PKCβII-specific inhibitor treatment. One week after PC-3 cell injection, mice were implanted with osmotic pumps with saline, control peptide (TAT) or βIIV5-3 conjugated to TAT at 3.6 mg/kg/day for 2 weeks followed by 36 mg/kg/day for the next 3 weeks. Deuterated water (4%) was administered for 1 week prior to sacrifice. (A) Tumor volume was measured weekly (repeated ANOVA, *; p<0.05, n=4–5 each). Tumors were excised and weighed at week 6. Final tumor weight was 40% lower in the βII V5-3-treated group but this difference did not reach statistical significance and there was no difference in body weight between the groups. (B) Five-week continuous βIIV5-3-treatment decreased PKCβII translocation to the particulate fraction of both tumors and livers. The active level of PKCβII was analyzed by Western blot after fractionation. GAPDH and Gαi were used as loading controls for the cytosolic (C) and particulate (P) fractions, respectively. IB; immunoblot. A 2-tailed Student’s t test was used to determine significance (n=3 each, p<0.05). (C) βIIV5-3 treatment in vivo results in reduced PKC kinase activity as measured in vitro, following immunoprecipitation with anti-PKCβII antibodies. Kinase assay was performed in the absence of added PKC activators, using histone (H3) as a substrate as described (30). The film was exposed for 3 days in −80C°. (D) A greater decrease in PC-3 tumor growth rate was obtained with a higher dose of βIIV5-3 (36 mg/kg/day for 4 weeks). A repeated ANOVA and a 2-tailed Student’s t test was used to determine significance (*; p<0.05 in repeated ANOVA, §; p<0.05 vs. TAT-treated and §§; p <0.005 vs. TAT-treated in t test, n=8–9 each, 16% vs. 60% reduction in the overall tumor growth rate, Figure 3A vs. 3D). Additional blots for (B) and (C) are provided in supplemental Figure 3.

Figure 4

Figure 4

Analysis of proliferation rates of tumor endothelial cells (TEC) and tumor cells (TC) after peptide treatment. (A) Mice treated with βIIV5-3 at 3.6 mg/kg/day for 2 weeks and with 36 mg/kg/day for the remaining 3 weeks were sacrificed at week 3 (mid point) and at the end of the treatment at week 6 and the proliferation rates of tumor endothelial cells were then determined after their isolation. Deuterated water was administered during the 7 days before sacrifice. A 2-tailed Student’s t test was used to determine significance (Figure 4A, _p_=0.008 at week 3, n=8–9 each). (B). Tumor sections from week 3 and 6 were stained with CD31-FITC antibodies and immunostaining intensity was quantified using Photoshop. A 2-tailed Student’s t test was used to determine significance (Figure 4B, week 3 data, *; p<0.05). Scale bar: 10 μm. (C) Mice treated with βIIV5-3 at 3.6 mg/kg/day for 2 weeks and with 36 mg/kg/day for the remaining 3 weeks were sacrificed at week 3 (mid point) and at the end of the treatment at week 6 to isolate and determine proliferation rates of tumor cells. A 2-tailed Student’s t test was used to determine significance (Figure 4C, _p_=0.0007 at week 3, n=8–9 each). (D) Tumor sections from week 3 and 6 were stained for TUNEL conjugated with Texas Red (Figure 4D, week 6 data, p=0.06, n=4). TUNEL staining was confirmed with cleaved caspase 3 staining (FITC- conjugated) of 3-week tumor samples (insert). Scale bars : 10 μm.

Figure 5

Figure 5

βIIV5-3 treatment reduced pericentrin levels and induced co-localization of PKCβII and pericentrin in PC-3 tumors. (A) The level of pericentrin was determined using tumor sections after a 4-week treatment with TAT or βIIV5-3 at 36 mg/kg/day. Sections were stained for pericentrin (Abcam, rabbit polyclonal Ab4448; followed by goat anti-rabbit conjugated to Cy3, pink) and for nuclei (Hoechst, blue). (B) The levels of both the 220 and 150kDa bands corresponding to pericentrin (arrows, (22, 44, 45)) were determined using total tumor lysates after a 4-week treatment with TAT or βIIV5-3 at 36 mg/kg/day. (C) Immunofluorescence staining of 4-week-treated tumors demonstrated co-localization of PKCβII and normal dot-structured pericentrin in βIIV5-3-treated tumors. Shown are nuclei staining (panels B1 and 5), PKCβII (panels B2 and 6, green), pericentrin (panels B3 and 7, red) and merged figure (panels B4 and 8). Arrows indicate co-localization of pericentrin and PKCβII (yellow), whereas asterisk shows filamentous pericentrin not co-localized with PKCβII. (D) The interaction of PKCβII and pericentrin was further confirmed by immunoprecipitation (IP). Immunoprecipitates from the detergent- solubilized total tumor lysate and PEC cultures using anti-PKCβII antibody were immunoblotted (IB) with the mixture of 4b, M1 and UM225 pericentrin antibodies (19) to detect pericentrin (Figure 5C, top panel, lanes 2 and 3, arrows). Both the 220 and 150kDa bands corresponding to pericentrin (22, 44, 45) were present in immunoprecipitates showing that they interact with PKCβII in vivo. The interaction with PKCβII was stronger with βIIV5-3 treatment (compare top panel, lanes 2 and 3). In the negative control (lane 1, incubated with IgG and immunoprecipitated with beads), the amount of pericentrin (top panel, lane 1) or PKCβII (lower panel, lane 1) present was not significant. Whole tumor lysates of tumor was used as a positive control (lane 4) to show pericentrin and PKCβII bands (upper and lower panels, lane 4). Also, immunoprecipitate from the lysate of primary culture of PECs that were not treated with βIIV5-3 was used as another control to show interaction of PKCβII and pericentrin (Lane 5).

Figure 6

Figure 6

Pericentrin abnormality is present in tumor endothelial cells and in human tumor endothelium. (A) Tumor sections from mice treated for 4 weeks with TAT or βIIV5-3 (36 mg/kg/day) were stained for nuclei (Hoechst), CD31 (green) and pericentrin (red). Scale bar: 10 μm. (B) The presence of abnormal pericentrin and centrosomal defects in tumor endothelial cells (TEC) grown in PC-3 conditioned medium was determined by immunofluorescence. Mouse tumor endothelial cells were grown in DMEM or in PC-3 conditioned medium (media from PC-3 cells grown for 2 days) and treated with TAT or βIIV5-3 at a final concentration of 1μM (added 3 times per day for 2 days). Representative images of TEC grown in DMEM with TAT (top), in PC-3 conditioned medium with TAT (middle) and with βIIV5-3 (bottom) are shown (representative of 3 experiments). Tumor endothelial cells were stained separately for pericentrin (green) and γ-tubulin (red). Merged figures are also shown (including nuclei stained with Hoechst). Scale bar: 10 μm. (C) Staining with anti-α-tubulin suggests abnormal microtubule structure in the tumor endothelial cells. Tumor endothelial cells treated the same as in (B) were stained separately for pericentrin (green) and α-tubulin (red). Merged figures are also shown (including nuclei staining with Hoechst). Scale bar: 10 μm. (D) The level of pericentrin is high in human prostate tumor endothelium. The level of pericentrin was determined using paraffin-embedded sections from human prostate with Gleason grades 3, 4 and 5 cancers and were stained for pericentrin and counterstained with hematoxylin. Representative pictures are shown (n=8; left: pericentrin staining on endothelium adjacent to benign prostatic hyperplasia, middle: pericentrin staining on tumor endothelium adjacent to tumor glands with Gleason grades 3+4, right: magnified view of the middle figure). Scale bars: 10 μm.

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