DNA licensing as a novel androgen receptor mediated therapeutic target for prostate cancer

Abstract

During middle G(1) of the cell cycle origins of replication orchestrate the ordered assembly of the pre-replication complex (pre-RC), allowing licensing of DNA required for DNA replication. Cyclin-dependent kinase activation of the pre-RC facilitates the recruitment of additional signaling factors, which triggers DNA unwinding and replication, while limiting such DNA replication to once and only once per cell cycle. For both the normal and malignant prostate, androgen is the major stimulator of cell proliferation and thus DNA replication. In both cases, the binding of androgen to the androgen receptor (AR) is required. However, the biochemical cascade involved in such AR-stimulated cell proliferation and DNA synthesis is dramatically different in normal versus malignant prostate cells. In normal prostate, AR-stimulated stromal cell paracrine secretion of andromedins stimulates DNA replication within prostatic epithelial cells, in which AR functions as a tumor suppressor gene by inducing proliferative quiescence and terminal differentiation. By direct contrast, nuclear AR in prostate cancer cells autonomously stimulates continuous growth via incorporation of AR into the pre-RC. Such a gain of function by AR-expressing prostate cancer cells requires that AR be efficiently degraded during mitosis since lack of such degradation leads to re-licensing problems, resulting in S-phase arrest during the subsequent cell cycle. Thus, acquisition of AR as part of the licensing complex for DNA replication represents a paradigm shift in how we view the role of AR in prostate cancer biology, and introduces a novel vulnerability in AR-expressing prostate cancer cells apt for therapeutic intervention.

Figure 1

Androgen receptor signaling in normal and malignant prostate cells. (top) In the normal prostate, growth and maintenance of prostatic epithelium depends on paracrine signaling of andromedins (growth and survival factors) produced by supporting stromal cells (smooth muscle and fibroblasts). Andromedins are secreted due to androgen signaling through AR, a nuclear hormone receptor expressed by prostate luminal epithelia but not by basal epithelia. (bottom) During prostate cancer transformation, the paracrine AR signaling mechanism is replaced by an emergent autocrine mechanism, whereby cancer cells exhibit less dependency on stromally-derived factors. Androgens acting through AR can directly stimulate production of growth and survival factors by the cancer cells.

Figure 2

Mammalian pre-RC formation and DNA licensing in the context of cell cycle progression. In early G₁, cyclin D-cdk4/6 phosphorylation of Rb, which permits E2F transcriptional activity, triggers entry into the cell cycle. Pre-RCs form via stepwise binding of licensing factors Orc1–6, Cdc6, Cdt1, and Mcm2–7, required for DNA licensing. Progressing into S-phase, CDK, and DDK phosphorylation of pre-RC constituents initiates Orc1 dissociation, and the removal and subsequent nuclear export of Cdc6, while geminin, along with CDK and DDK activity, trigger the removal and degradation of Cdt1. This regulation serves to ‘un-license’ the DNA and prevents re-licensing before completion of the initiated cell cycle. E2F target genes: Cdc6, MCM, Dbf4 (DDK), Cyclins E, and A. SCF^SKP2, Skp1-cullin-F box E3 ubiquitin ligase; CUL4, CUL4-DDB1-CDT2 ubiquitin ligase; R, restriction point; G, geminin; U, ubiquitin; P, phosphate.

Figure 3

AR interacts with members of the DNA licensing complex. Androgen-sensitive LNCaP prostate cancer cells express AR as well as members of the DNA licensing complex, Orc2, Cdt1, and Cdc6 (lane 1). Immunoprecipitation (lane 2 – IgG control, lane 3 – anti-AR antibody) of AR from LNCaP cells followed by western blotting reveal that Orc2, Cdt1, and Cdc6 co-immunoprecipitated with AR. These data demonstrate AR presence at pre-RCs and strongly support a role for AR in modulating DNA replication licensing to drive prostate cancer cell proliferation. Figure from Vander Griend & Isaacs (2008) (LAPC4 data not shown).