Loss of Androgen Receptor-Dependent Growth Suppression by Prostate Cancer Cells Can Occur Independently from Acquiring Oncogenic Addiction to Androgen Receptor Signaling

The conversion of androgen receptor (AR) signaling as a mechanism of growth suppression of normal prostate epithelial cells to that of growth stimulation in prostate cancer cells is often associated with AR mutation, amplification and over-expression. Thus, down-regulation of AR signaling is commonly therapeutic for prostate cancer. The E006AA cell line was established from a hormone naïve, localized prostate cancer. E006AA cells are genetically aneuploid and grow equally well when xenografted into either intact or castrated male NOG but not nude mice. These cells exhibit: 1) X chromosome duplication and AR gene amplification, although paradoxically not coupled with increased AR expression, and 2) somatic, dominant-negative Serine-599-Glycine loss-of-function mutation within the dimerization surface of the DNA binding domain of the AR gene. No effect on the growth of E006AA cells is observed using targeted knockdown of endogenous mutant AR, ectopic expression of wild-type AR, or treatment with androgens or anti-androgens. E006AA cells represent a prototype for a newly identified subtype of prostate cancer cells that exhibit a dominant-negative AR loss-of-function in a hormonally naïve patient. Such loss-of-function eliminates AR-mediated growth suppression normally induced by normal physiological levels of androgens, thus producing a selective growth advantage for these malignant cells in hormonally naïve patients. These data highlight that loss of AR-mediated growth suppression is an independent process, and that, without additional changes, is insufficient for acquiring oncogene addiction to AR signaling. Thus, patients with prostate cancer cells harboring such AR loss-of-function mutations will not benefit from aggressive hormone or anti-AR therapies even though they express AR protein

Loss of Androgen Receptor-Dependent Growth Suppression by Prostate Cancer Cells Can Occur Independently from Acquiring Oncogenic Addiction to Androgen Receptor Signaling

The conversion of androgen receptor (AR) signaling as a mechanism of growth suppression of normal prostate epithelial cells to that of growth stimulation in prostate cancer cells is often associated with AR mutation, amplification and over-expression. Thus, down-regulation of AR signaling is commonly therapeutic for prostate cancer. The E006AA cell line was established from a hormone naïve, localized prostate cancer. E006AA cells are genetically aneuploid and grow equally well when xenografted into either intact or castrated male NOG but not nude mice. These cells exhibit: 1) X chromosome duplication and AR gene amplification, although paradoxically not coupled with increased AR expression, and 2) somatic, dominant-negative Serine-599-Glycine loss-of-function mutation within the dimerization surface of the DNA binding domain of the AR gene. No effect on the growth of E006AA cells is observed using targeted knockdown of endogenous mutant AR, ectopic expression of wild-type AR, or treatment with androgens or anti-androgens. E006AA cells represent a prototype for a newly identified subtype of prostate cancer cells that exhibit a dominant-negative AR loss-of-function in a hormonally naïve patient. Such loss-of-function eliminates AR-mediated growth suppression normally induced by normal physiological levels of androgens, thus producing a selective growth advantage for these malignant cells in hormonally naïve patients. These data highlight that loss of AR-mediated growth suppression is an independent process, and that, without additional changes, is insufficient for acquiring oncogene addiction to AR signaling. Thus, patients with prostate cancer cells harboring such AR loss-of-function mutations will not benefit from aggressive hormone or anti-AR therapies even though they express AR protein.</p

AR in E006AA cells undergoes nuclear translocation, binds DNA but is not transcriptionally activity due to the S599G loss-of-function mutation.

<p>(A) Western blot analysis of nuclear and cytoplasmic AR expression in LNCaP C4-2B, PC-3, and E006AA cells, treated with or without androgen. E006AA fractions were blotted for Vinculin and HDAC to confirm suitable cytoplasmic and nuclear separation, respectively. (B) PCR amplification of a putative ARE in the PSA promoter region from anti-AR immunoprecipitated chromatin from LNCaP C4-2B and E006AA cells, treated with or without androgen. Chromatin immunoprecipitated with an IgG isotype served as a negative control. (C) Quantitative PCR analysis of anti-AR antibody immunoprecipitated chromatin from LNCaP C4-2B and E006AA cells, treated with or without androgen: (Ci) PSA promoter ARE amplification in C4-2B cells; (Cii) PSA enhancer ARE III amplification in C4-2B cells; (Ciii) PSA promoter ARE amplification in E006AA cells; (Civ) PSA enhancer ARE III amplification in E006AA cells. The results are presented as percentage of input. (*, p-value <0.05, Student's T-test). (D) Analysis of AR transcriptional activity in E006AA cells compared to LNCaP C4-2B, PC-3, E006AA Lenti-AR, E006AA Lenti-non-silencing-shRNA, E006AA Lenti-AR-shRNA cells, treated with or without androgen. (E) Introduction of mutant S599G AR into androgen-responsive, PSA producing LNCaP C4-2B cells, and its effect on AR-driven PSA-Luciferase activity in C4-2B cells, with or without androgen (*, p-value <0.05, Student's T-test). All experiments in this section were performed with cells grown in 10% csFBS supplemented media, +/−24 hr stimulation with 1.0 nM R1881.</p

In vitro growth response to modulation of AR expression in E006AA cells.

<p>(A) Cell growth analyses of E006AA cells transduced with or without Lentiviral wild-type AR, which were then treated with 10 µM Casodex or an increasing dose of R1881 (1.0 nM to 100 nM). Cell growth is graphed relative to the respective untreated control cells. (B) Comparison of vector only Lentiviral-non-silencing, Lenti-AR, and Lenti-AR-shRNA transduced E006AA cell growth relative to control cells. (C) Colony forming assay comparing parental control cells to non-silencing, and two different anti-AR Lenti-shRNA sequences (3715 and 3718).</p

E006AA cells are tumorigenic and immunogenic in nude mice, and do not require a physiologic level of testosterone for growth.

<p>(A) Inoculation of E006AA cells into male nude mice compared to male NOG-SCID mice. (B) Transfer of tumor tissue derived from an established E006AA xenograft tumor from a male NOG-SCID mouse to intact male Nude and NOG-SCID mice. (C) The growth of E006AA and CWR22 human prostate cancer xenograft tumors in castrated vs. intact male mice at eight weeks. (D) Histological analysis (H&E) and immunostaining of E006AA xenograft tissue sections for AR, PSA, p63, and CK5 (images taken at 20× and 40×). (E) Western blot analysis of CK8 and CK18 expression in LNCaP, PC-3, E006AA and S006AA cells. ß-Actin served as a loading control.</p

Mesenchymal stem cell infiltration during neoplastic transformation of the human prostate.

Mesenchymal Stem Cells (MSCs) have been identified in prostate cancer, raising the critical question of their physical and temporal source. Therefore, MSCs were quantified and characterized in benign and malignant prostate tissue representing different disease states and a wide range of age groups from fetal development through adult death using analytical and functional methodologies. In contrast to lineage-restricted Mesenchymal Progenitor Cells (MPCs) found in normal prostate tissue, MSCs with tri-lineage differentiation potential (adipogenesis, osteogenesis, and chondrogenesis) are identified in prostate tissue from a subset of men with prostate cancer, consistent with an influx of more stem-like progenitors (i.e. MSCs) from the bone marrow. Additionally, prostate tissue from a subset of these patients is highly enriched in MSCs, suggesting their enumeration may have prognostic value for identifying men with aggressive disease. This influx is an ongoing process continuing throughout disease progression as documented by the presence of MSCs in metastatic lesions from multiple organ sites harvested at the time of death in metastatic castration-resistant prostate cancer (mCRPC) patients. This infiltration of MSCs from systemic circulation provides the rationale for their use as a cell-based vector to deliver therapeutic agents

Somatic AR mutation and amplification in E006AA cells.

<p>(A) Cytogenetic analysis of AR status in normal male control and E006AA cells as determined by Fluorescence in Situ Hybridization (FISH) using probes specific for the AR Gene (Xq12, red colored) and the X centromere (green colored). (B) Sequencing results of overlapping primer PCR amplification of E006AA mRNA and cDNA identified a missense mutation (Ser599Gly), which corresponds to the “X” position of the conserved AXRND motif in the DBD D-box (as denoted by black arrowhead at position 597 of the reference human AR NM_000044), as found in human AR, GR, PR, and MR. (C) Quantitative PCR analysis of AR mRNA transcript levels in LNCaP compared to E006AA cells. (D) Western blot analysis of AR protein expression in E006AA cells infected with lentiviral non-silencing shRNA, wild-type AR (Lenti-AR), or anti-AR shRNA (AR shRNA) constructs compared to parental E006AA, LNCaP, PC-3 and S006AA stromal cells. Protein lysates were loaded on a per cell basis as follows: LNCaP (20,000), all others (200,000) cells per lane.</p

Karyotype Analysis of E006AA cells.

<p>(A) Karyotype analysis of E006AA cells prior to injection and <i>in vivo</i> tumor growth. (B) Analysis of E006AA cells derived from a NOG-SCID tumor-bearing mouse, documenting no significant karyotypic alterations during tumor growth.</p