Haploinsufficiency of the maspin tumor suppressor gene leads to hyperplastic lesions in prostate

Abstract

Maspin is a key tumor suppressor gene in prostate and breast cancers with diverse biological functions. However, how maspin regulates prostate tumor progression is not fully understood. In this study, we have used maspin heterozygous knockout mice to determine the effect of maspin haploinsufficiency on prostate development and tumor progression. We report that loss of one copy of maspin gene in Mp(+/-) heterozygous knockout mice leads to the development of prostate hyperplastic lesions, and this effect was mediated through decreased level of cyclin-dependent kinase inhibitors p21 and p27. Prostate hyperplastic lesions in Mp(+/-) mice also induced stromal reaction, which occurred in both aged prostate tissues and in neonatal prostates during early ductal morphogenesis. We showed that maspin was also expressed in prostate smooth muscle cells (PSMC), and recombinant maspin increased PSMC cell adhesion but inhibited cell proliferation. We also observed a defective interaction between epithelial cells and basement membrane in the prostate of Mp(+/-) mice, which was accompanied with a changed pattern of matrix deposition and a loss of epithelial cell polarity. Therefore, we have identified a novel property of maspin, which involves the control of the proliferation in prostate epithelial and smooth muscle cells. This is the first report that a partial loss of maspin caused an early developmental defect of the prostate and prostate hyperplastic lesions in mouse.

Fig .1

Maspin expression in mouse prostate. Panel A, maspin expression analyzed by immunostaining in 24 week-old ventral prostate (VP) of Mp^+/+ wildtype mouse. Panel B, analysis of maspin expression at mRNA level by RT-PCR. VP tissues were harvested from wildtype Mp^+/+ and maspin heterozygous mice (Mp^+/-) at 24 wks. Lower panel, relative signals of maspin mRNA quantitated by NIH J image software. Panel C, IP-Western blot analysis of maspin protein level with protein extracts from various samples. Tubulin serves as a loading control. Note that maspin level was reduced at mRNA and protein levels in Mp^+/- VPs (B, C). Lower panel, relative signals of maspin protein quantitated by NIH J image software. ** indicates p value < 0.01 (t-Test).

Fig. 2

Development of prostate hyperplastic lesions in Mp^+/- heterozygous mice. Upper panels, representative views of the histology of dorsal prostate tissues (DPs) from wildtype (WT) and Mp^+/- mice at 6 and 12 months of age. Note the lack of significant stroma surrounding the glands in WT 6 month-old DP. The epithelium is relatively flat with few infoldings. DPs from Mp^+/- mice showed the presence of hyperproliferative, multiple layers of epithelial cells, with the thickened stroma surrounding the gland and the epithelial hyperplasia. The incipient stromal proliferation inside the epithelial folding became more prominent in Mp^+/- 12 month DP. Lower panels, immunostaining of sections the upper panels with anti-SMA (α-smooth muscle actin). Note the increased multi-layers of stromal cells in 12 month-old Mp^+/- DP.

Fig. 3

Mp^+/- neonatal mice developed a defect in early ductal morphogenesis and changed stromal patterns in the ventral prostates. Panel A left, microscopic view of ductal growth in the dissected VPs from day 8 neonatal mice. Arrows indicate the nodes. WT, wildtype day 8-old VP. Mp^+/-, Mp^+/- day 8-old VP. Panel A right, comparison of node numbers in WT and Mp^+/- VPs. Panel B, Histology and immunohistochemistry of WT and Mp^+/- VPs. Note that Mp^+/- VPs showed elevated epithelial and stroma proliferation. Double immunostaining of α-smooth muscle actin (SMA) and keratin-8 (CK-8) were performed with anti-SMA and anti-CK-8 antibodies, using samples from 8-day-old VPs isolated from WT and Mp^+/- mice. Red signals indicated α-smooth muscle actin positive cells, and green signals indicated keratin-8 positive basal epithelial cells. Slides were counterstained with DAPI to indicate the cell nuclei. Bars, 50 μM.

Fig. 4

Maspin inhibits prostate epithelia cell proliferation. Panel A, quantitative analyses of prostate epithelial cell proliferation rate in WT and Mp^+/- VPs and APs. Samples at designated ages were stained with an anti-Ki-67 antibody. Left, AP and VP from 6-8 month-old mice (N=10 for both WT and Mp^+/- groups). Right, AP and VP from and 12-15 month-old mice (N=5 for both Mp^+/+ and Mp^+/- groups). *, P value < 0.05; **, P value < 0.01. Panel B, cell cycle analyses of prostate C2N and maspin-expressing C2N-Mp cells by flow cytometry. Cells were either asynchronized or synchronized as indicated. Cell synchronization was performed with mimosine (see Material and Method), and cells were released, and cultured in mimosine-free medium for six hours before being harvested for cell cycle analysis by flow cytometry. Panel C, analysis of cyclin E-associated Cdk2 kinase activity. Cell extracts were immunoprecipitated with antibodies against cyclin E or Cdk2. Cdk2 kinase activity was assayed using histone H1 as a substrate, and kinase activities were quantitated using assay blots and NIH J image software (right panel). Bars were from triplicate samples. Cyclin E and Cdk2 served as IP controls, and actin served as sample loading control. D. Analysis of p21 and p27 levels in C2N cells and in dorsal prostate (DPs) from WT and Mp+/- mice. Left, C2N and C2N-Mp cell extracts (from panel C) were immunoprecipitated with anti-cyclin E or anti-cdk2, and blotted for p21 and p27 with anti-p21 or anti-p27 antibodies. Note the increased levels of p21 and p27 in C2N-Mp cells. Cyclin E and Cdk2 served as IP controls, and actin served as sample loading control. Right, quantitation of immunohistochemical analyses for the percentage of p21- and p27-positive epithelial cells in WT and Mp^+/- prostate. DPs from WT and Mp^+/- mice (6-8 month-old) were stained with antibodies against p21 and p27. A total of 3,000 epithelial cells were counted for each sample. Bars were from 5 mice at 6- to 8-month of age. ** indicates p value < 0.05, *** indicates p value < 0.01.

Fig. 5

Evidence that maspin is expressed in smooth muscle cells and maspin increases smooth muscle cell adhesion. A. Western blot analysis of cell extracts from prostate smooth muscle cells and NIH3T3 cells. B. Prostate smooth muscle cell proliferation assay. PSMC cells were treated with GST or GST-Mp daily at 0.5 μM for three days and cell numbers were measured as described in the Materials and Methods. Bars were from three assays. P value < 0.05. C. PSMC cells were pre-incubated with GST or GST-maspin at the indicated concentrations (μM) for 30 minutes. 2 × 10⁴ cells/well were plated on FN matrix, and cell adhesion was quantified as described in Materials and Methods. Control indicates PSMC cells that were mock-treated with PBS buffer. Data were from three assays. Statistical analysis was done by a t-test. ** indicates p value <0.05 compared to control sample.

Fig. 6

Mp^+/- mice exhibit altered patterns of matrix protein production and cell-cell interactions in prostate. H & E, histology of WT and Mp^+/- DP samples (12 months) by H & E staining. Insets, higher power view of a selected area from WT and Mp^+/- DPs. LN, immunostaining of prostate DPs with an antibody against laminin-1 (LN). Anti-LN antibody was recognized by a FITC-conjugated secondary antibody. Blue, counterstaining with DAPI to indicate cell nuclei. Insets, selected regions with higher magnification. E-Cad & ZO-1, patterns of E-cadherin and ZO-1 expression analyzed by double immunostaining using WT and Mp+/- DPs. Double immunostaining and the use of Texas-red and FITC-conjugated secondary antibodies were described in the Material and Methods. Red regions indicate E-cadherin immunoreactivity, and green regions indicate ZO-1 immunoreactivity (see arrows). Sections were counterstained with the DAPI to indicate cell nuclei. Bars, 50 μM.