Wnt signaling prevents prostatic epithelial cell differentiation. (A,B,C) p63 immunocytochemistry (red) of the P2 rat ventral prostate organ cultures maintained for 7 days in the absence (A) or presence of 50 nM of Wnt3a (B) or 400 nM of DKK1 (C).

<p>The tissue sections were counterstained with DAPI (blue). While p63 positive cells (purple) represent basal cells where progenitor cells reside, the blue cells (arrows) that are negative to p63 in the epithelium are differentiated luminal cells. (D) Quantification of p63 positive cells over total epithelial cells. Data were collected from randomly selected 22–27 ductal units from sections of the organ cultures per group and are expressed as mean + SEM (t-test). Note that while Wnt3a led to a significant increase in the number of basal cells, DKK1 resulted in a reduction in basal cells. Bar, 50 µm for A-C.</p

TaqMan RT-PCR analyses of Axin2 expression during prostate development and regrowth following androgen deprivation and replacement.

<p>(A) A gradual downregulation of Axin2 from newborn to adulthood.nbsp;(B) Axin2 was upregulated following castration, but returned to normal low levels after androgen replacement. Data were collected from 4–6 samples per group and are expressed as mean+SEM (t-test, compared to normal adult prostates). Abbreviation: N, normal prostate; C3, 3 days after castration, C17; 17 days after castration; C14+T. 14 days after castration+3 days of testosterone treatment.</p

Activation of Wnt signaling enhances prostate epithelial cell proliferation.

<p>(A-F) Shown are anti-BrdU antibody (B,D,F) and DAPI (A,C,E) double labeling of the P3 rat ventral prostate organ cultures maintained for 3 days in the absence (A,B) or presence of 50 nM of Wnt3a (C,D) or 400 nM of DKK1(E,F). (G). Quantification of BrdU-positive cells in a given visual field of 434 µm×322 µm. (H) Quantification of Ki67-positive cells in the P2 prostate cultures maintained for 7 days, which was normalized to the control cultures. Cell counts (for G and H) were performed from randomly selected visual fields of 5 organ cultures per group and data were expressed as mean+SEM (t-test). Note that while Wnt3a significantly enhanced progenitor cell proliferation, DKK1 inhibited progenitor cell proliferation. (I). Expression level change of cyclin B2 in P2 rat prostate organ cultures.. Data were collected from 4 organ cultures maintained for 2 days per group and are expressed as mean+SEM (t-test). Note that expression of cyclin B2 was upregulated by wnt3a, but down-regulated by DKK1. Bar, 100 µm for A-F.</p

DKK1 inhibits proliferation and migration of prostate cancer cells.

<p>(A) Tritiated thymidine incorporation in PC3 cultures in the absence or presence of Wnt3a or DKK1. While high concentration of Wnt3a (10nM) enhanced PC3 cell proliferation, DKK1 inhibited PC3 proliferation in a dose-dependent manner. (B) Regulation of PC3 cell migration by Wnt signaling. Data were collected from 6–8 cultures per group and are expressed as mean+SEM (t-test). Note that Wnt3a increased the number of migrated cells, whereas DKK1 inhibited cell migration.</p

Wnt signaling is activated in prostate cancer cells.

<p>TaqMan RT-PCR analysis of Axin2 expression in various human prostate epithelial cells and cancer cells. Data were collected from triplets and are expressed as mean+SEM (t-test). Note that Axin2 expression is much higher in prostate cancer cell lines (LNCaP, Du145, PC3) and human prostate tumor xenografts (LuCaP35, LuCaP77) as compared to primary prostate epithelial cells (PrEC) or non-tumorigenenic immortalized prostate epithelial cells (BPH1).</p

Wnt3a and DKK1 regulate prostatic epithelial branching morphogenesis.

<p>Whole mount ventral prostates were prepared from P2 rats and maintained for 7 days in serum-free medium in the absence (A) or presence of 50 nM of Wnt3a (B) or 400 nM of DKK1 (C). Similar patterns were consistently seen in 3 repeat experiments of 5–6 prostate organs per group in each individual experiment. Note that addition of either Wnt3a or DKK1 to the culture resulted a change in epithelial branching morphogenesis. Quantification of the cultures by measuring the diameter of the cultured prostates (D) and the ductal tips (E) using Axiovision software and the branching points (F) were done by analyzing 4 randomly selected cultures per group. Data are expressed as mean + SEM (t-test, compared to control cultures). Bar, 400 µm.</p

Mutations in the Hedgehog Pathway Genes <em>SMO</em> and <em>PTCH1</em> in Human Gastric Tumors

<div><p>The causal role of the hedgehog pathway in cancer has been best documented in basal cell carcinoma of the skin. To assess potential DNA alterations of the hedgehog pathway in gastric cancer, we sequenced <em>SMO</em> and <em>PTCH1</em> genes in a set of 39 gastric tumors. Tumors were classified by histology based on the Lauren classification and Sanger sequencing was performed to obtain full length coding sequences. Genomic instability was evident in these tumors as a number of silent or missense mutations were found. In addition to those that are potential germline polymorphisms, we found three <em>SMO</em> missense mutations, and one <em>PTCH1</em> frameshift mutation that are novel and have not been documented in basal cell carcinoma. Mutations were found in both intestinal and diffuse type gastric tumors as well as in tumors that exhibit both intestinal and diffuse features. mRNA expression of hedgehog pathway genes was also examined and their levels do not indicate unequivocal higher pathway activity in tumors with mutations than those without. In summary, <em>SMO</em> and/or <em>PTCH1</em> mutations are present at low frequency in different histologic subtypes of gastric tumors and these do not appear to be driver mutations.</p> </div

<b>Table 1.</b><i>SMO</i> and <i>PTCH1</i> Mutations in 39 Gastric Tumors.

<p><b>Table 1.</b><i>SMO</i> and <i>PTCH1</i> Mutations in 39 Gastric Tumors.</p

Representative H&E staining images for histopathologic assessment of gastric tumors.

<p>(A) A moderately differentiated intestinal type adenocarcinoma. Note the glandular pattern of neoplastic growth, tumor cells with scant cytoplasm and conspicuous nucleoli, and the fibrous tissue associated with lymphocytic infiltrate between glands. (B) An adenocarcinoma of diffuse type. Note single cells with signet ring features infiltrating the gastric wall. (images are at 40× magnification).</p

Expression of Gli3 and PTCH1 mRNA in gastric tumors with <i>PTCH1</i> mutations.

<p>Scatter plots for Gli3 and PTCH1, two hedgehog pathway down-stream genes expressed at relatively higher levels are shown according to their <i>PTCH1</i> mutation status. Expression values have been RMA normalized and presented as log value. Note the relative lower level of Gli3 and PTCH1 expression in the tumor with R1307fs mutation.</p