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. 2017 May 17;8(46):80265-80277.
doi: 10.18632/oncotarget.17919. eCollection 2017 Oct 6.

Deletion of tumor suppressors adenomatous polyposis coli and Smad4 in murine luminal epithelial cells causes invasive prostate cancer and loss of androgen receptor expression

Kenneth C Valkenburg  1 , Angelo M De Marzo  2   3 , Bart O Williams  1
Affiliations

Deletion of tumor suppressors adenomatous polyposis coli and Smad4 in murine luminal epithelial cells causes invasive prostate cancer and loss of androgen receptor expression

Kenneth C Valkenburg et al. Oncotarget. .

Abstract

Prostate cancer is the most diagnosed non-skin cancer in the US and kills approximately 27,000 men per year in the US. Additional genetic mouse models are needed that recapitulate the heterogeneous nature of human prostate cancer. The Wnt/beta-catenin signaling pathway is important for human prostate tumorigenesis and metastasis, and also drives tumorigenesis in mouse models. Loss of Smad4 has also been found in human prostate cancer and drives tumorigenesis and metastasis when coupled with other genetic aberrations in mouse models. In this work, we concurrently deleted Smad4 and the tumor suppressor and endogenous Wnt/beta-catenin inhibitor adenomatous polyposis coli (Apc) in luminal prostate cells in mice. This double conditional knockout model produced invasive castration-resistant prostate carcinoma with no evidence of metastasis. We observed mixed differentiation phenotypes, including basaloid and squamous differentiation. Interestingly, tumor cells in this model commonly lose androgen receptor expression. In addition, tumors disappear in these mice during androgen cycling (castration followed by testosterone reintroduction). These mice model non-metastatic castration resistant prostate cancer and should provide novel information for tumors that have genetic aberrations in the Wnt pathway or Smad4.

Keywords: Apc; basaloid; mouse model; prostate cancer; squamous.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors have no financial conflicts of interest to disclose.

Figures

Figure 1
Figure 1. Rationale behind the Apc-Smad4 double knockout (ApccKOSmad4cKO) mouse model
(A) Smad4 immuno-staining on prostate tissue from Apcflox and ApccKO mice (scale bars = 100 μm); (B) Axin2 and Smad4 quantitative reverse-transcriptase PCR from Apcflox and ApccKO prostate tissue (error bars represent standard deviation based on technical triplicates; ***P value < 1 × 10−4); (D) allele-specific PCR for Apc and Smad4 showing the floxed band (flox) and the band as a result of deletion (del) in prostate and bulbourethral gland tissue; and (C) timeline of tamoxifen administration and sacrifice of control and experimental mice.
Figure 2
Figure 2. Proliferative increases in ApccKOSmad4cKO mice compared to ApccKO mice
(A) Urogenital tracts, including prostate tissue, from ApcfloxSmad4flox, ApccKO, and ApccKOSmad4cKO mice (arrows indicate the anterior prostate lobes); (B) H&E staining and Ki67 immuno-staining of prostate tissues (scale bars = 100 μm); and (C) Nuance microscope quantification of proliferating (Ki67-positive) cells in prostate cells from ApcfloxSmad4flox, ApccKO, and ApccKOSmad4cKO mice. For all groups, P value < 1.0 ×10−12.
Figure 3
Figure 3. Gain of beta-catenin expression plus loss of Smad4 expression is associated with increased proliferation
Benign and malignant prostate tissue from ApccKOSmad4cKO mice were stained for hematoxylin and eosin (H&E) and immuno-stained for beta-catenin, Smad4, Ki67, and cyclin D1. Arrows point to regions keratohyalin granule formation and loss of nuclei (H&E stain) and regions of nuclear beta-catenin staining. Scale bars = 50 μm.
Figure 4
Figure 4. Concurrent loss of AR and luminal marker CK8 and expansion of basal markers CK14 and p63 in malignant tissue
Benign and malignant prostate tissue from ApccKOSmad4cKO mice were immuno-stained for androgen receptor (AR), cytokeratin 8 (CK8), cytokeratin 14 (CK14), and p63. Arrows point to representative positively stained CK14 and p63 basal cells. Scale bars = 50 μm.
Figure 5
Figure 5. Mix of differentiation phenotypes in ApccKOSmad4cKO tumors
Representative hematoxylin and eosin staining of prostate tumors from ApccKOSmad4cKO mice. Squamous in situ carcinoma (A–B); scale bars = 100 μm (A) and 200 μm (B). Squamous carcinoma with muscular invasion (C–D); scale bars = 250 μm (C) and 25 μm (D). Basaloid carcinoma (E); scale bar = 50 μm. Representative image of mixed basaloid and squamous cell carcinoma in the same region (F); scale bar = 50 μm. Representative images of basaloid region of prostate tumor stained with beta-catenin (G) and Ki67 (H); scale bars = 50 μm.
Figure 6
Figure 6. Tumors in ApccKOSmad4cKO mice are castration resistant
(A) Timeline of tamoxifen administration, castration, and sacrifice of control and experimental mice; and (B) representative images of prostate tissue from ApcfloxSmad4flox, ApccKO, and ApccKOSmad4cKO mice were stained for hematoxylin and eosin (H&E) and immuno-stained for beta-catenin and Ki67. Scale bars = 100 μm.
Figure 7
Figure 7. Loss of cancer phenotype after one or more rounds of androgen cycling
Timelines of one rounds of androgen cycling (A) and two rounds of androgen cycling (C) in control and experimental mice. Representative hematoxylin and eosin (H&E), beta-catenin, and Ki67 staining of prostate tissue from ApcfloxSmad4flox and ApccKOSmad4cKO mice (B and D). Scale bars = 100 μm.
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