**1. Introduction**

34 Sex Steroids

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Sex hormone-binding globulin (SHBG) is a sex steroid binding protein, originally described in humans as the major binding protein for estrogens and androgens in plasma (Anderson, 1974; Avvakumov, et al, 2010). By governing equilibrium conditions in plasma between bound and free sex steroids, SHBG regulates the availability of the latter to hormonally responsive tissues. Along with regulating free steroid concentrations in plasma, it is increasingly evident that SHBG also participates in other biological processes. These include, but are not limited to- activation of a rapid, membrane based steroid signaling pathway in tissues such as the prostate and breast (Rosner et al, 2010); spermatogenesis (Selva and Hammond, 2006); and a yet to be determined consequence of co-localization with oxytosin in brain cells (Caldwell et al, 2006).

Plasma based SHBG is extensively studied, especially in the context of its regulation of free steroid concentrations and epidemiologic associations. The origin of plasma SHBG is, for all intents and purposes, the liver (Khan et al, 1981; Pugeat et al, 2010) (a differentially glycosylated isoform, androgen binding protein (ABP) is synthesized in the testis (Vigersky et al, 1976)). However, we now know that SHBG is also synthesized, albeit to a much lesser degree, in certain hormonally responsive tissues (Kahn et al, 2002). Early studies demonstrated immunoreactive SHBG in the prostate and breast (Bordin & Petra 1980; Tardivel-Lacombe et al, 1984; Sinnecker et al, 1988; 1990; Meyer et al, 1994; Germain et al, 1997), though its origin (local synthesis vs. import from plasma) was unclear. Other studies demonstrated SHBG mRNA in certain nonhepatic tissues (Larrea et al, 1993; Misao et al, 1994; 1997; Moore et al, 1996; Murayama et al, 1999), and one reported both SHBG protein and mRNA together in fallopian tube tissue (Noé, 1999). In 2002, we reported that human prostate tissue expresses both SHBG mRNA and protein, as do prostate cancer cell lines (Hryb et al, 2002), suggesting that SHBG is indeed locally

<sup>\*</sup> Corresponding Author

Sex Hormone-Binding Globulin as a Modulator of the Prostate "Androgenome" 37

Our analyses also included a detailed look at the SHBG expression patterns in normal human prostate tissue and the LNCaP prostate cancer cell line (Nakhla et al, 2009). Focusing on PL, we found that only the eight exon long SHBG transcript is generated in normal prostate tissue. This suggests that alternatively spliced PL–derived species are either not present, that they exist at levels undetectable by our RT-PCR assay, or that they are synthesized in minor cellular populations within normal prostate tissue. Compared to normal liver tissue, quantitative PCR analysis revealed that normal prostate expresses only 1/1000th the abundance of total PL-derived transcripts. Even taking into account the relative complexity of the PL-transcript expression pattern in normal liver, with the SHBG transcript being most abundant, these findings are in concordance with hepatic SHBG being synthesized for global use (plasma), and prostate SHBG being synthesized for local, or intracellular use. Normal prostate revealed a low abundance of transcripts derived from the two upstream promoters we examined. In striking comparison, the LNCaP prostate cancer cell line exhibited a dramatic relative increase in both the number of alternatively spliced transcripts and transcripts from upstream promoters. The reasons behind these differences in SHBG gene transcription profiles are unclear, they could reflect the clonality of LNCaP cells vs. whole prostate tissue, dysregulation of global RNA processing in LNCaP, and/or changes in specific SHBG mRNA processing elements, among other possibilities. Taken together, the SHBG gene may be a valuable provider of diagnostic, prognostic, and

Because SHBG binds androgens, we hypothesized that a major function of locally expressed SHBG in prostate cells might be to regulate the androgenome. We set out to investigate two different scenarios by which SHBG could influence androgen signaling. First was that locally synthesized SHBG could modulate the binding of androgen to the androgen receptor (AR) by acting as a steroid sequestering agent. For example, in the same way that plasma SHBG regulates the concentrations of plasma free steroids, intracellular SHBG could regulate intraprostatic free testosterone and dihydrotestosterone (DHT). Perhaps relevant to prostate cancer progression, this model predicts that diminished intracellular SHBG would allow for increased free intracellular DHT and hence increase the effect of intracellular androgens. The second scenario envisions that locally expressed SHBG can participate, in an autocrine/paracrine manner, in a rapid, membrane based signaling pathway in prostate cells (Kahn et al, 2002; Kahn et al, 2003; Rosner et al, 2010). The initial steps of this pathway are well established biochemically, however little is understood about its biologic functions. Briefly, SHBG, in its steroid-free configuration, binds to a high affinity, but yet to be cloned membrane receptor (RSHBG), forming a bipartite complex (SHBG-RSHBG). Subsequently, DHT binds to and activates the SHBG-RSHBG complex causing a rapid induction of cAMP and the

We developed a functional microarray approach to ascertain the effects of SHBG on the androgenome of LNCaP cells (Kahn et al, 2008). Using an inducible system that enabled

**2.3 SHBG expression in normal prostate tissue and the LNCaP prostate cancer** 

predictive biomarkers for individuals with prostate cancer.

**3. SHBG and its effects on the prostate "androgenome"** 

activation of protein kinase A. This occurs independently of the AR.

**3.2 Functional microarray analysis** 

**cell line** 

**3.1 Introduction** 

expressed by prostate cells. We therefore set out to ascertain the biological functions associated with locally expressed SHBG in the prostate. High on our list was that locally expressed SHBG could regulate the prostate cellular response to androgen signaling by modulating the expression of androgen responsive genes, referred to herein as the "androgenome".

In this chapter, we first present an overview of human SHBG gene expression, as recent studies from our group and Pinós et al, have shown it to be far more complex than previously thought. We then review our work on the effects of SHBG on the prostate androgenome, along with our most recent findings on how SHBG modulates the expression of specific and noteworthy androgen receptor (AR) responsive genes. We conclude by addressing how SHBG, through its effects on the androgenome, might affect prostate biology, and how altered SHBG expression may influence prostate cancer progression.

#### **2. SHBG gene structure and expression**

#### **2.1 Introduction**

In plasma, SHBG exists as a homodimer, whose subunits are derived from an eight-exon long transcript as a 402 amino acid precursor protein that is glycosylated (sometimes differentially) and cleaved at its amino terminus to remove a 29 amino acid signal peptide (Hammond et al, 1987; Gershagen et al, 1989; Joseph, 1994; Avvakumov et al, 2010). The same eight-exon long transcript also encodes androgen binding protein (ABP) in the testis, an alternatively glycosylated form of SHBG (not a topic of this review). The human SHBG gene is located on chromosome 17p13.1, ~30Kb from the p53 tumor suppressor gene. As a result, in most instances where hemizygous deletions of this oft-targeted chromosomal region occur in prostate tumors, it is likely that DNA sequences involving both genes are lost.

#### **2.2 The human SHBG gene transcription pattern.**

Bolstered by recent reports (Nakhla et al, 2009; Pinós et al, 2009), we now know that transcription of the human SHBG gene is highly complex, as well as tissue dependent. The eight-exon long SHBG transcript is derived from a downstream promoter, designated here as PL. In addition to the SHBG transcript, we found that at least five different mRNA species are generated through alternative splicing of exons 4-7 from the primary PL derived transcript (Nakhla et al, 2009). Adding to the overall complexity of human SHBG gene transcription, we and others have detected at least five independent first exons in novel SHBG gene transcripts (Gershagen et al, 1989; Nakhla et al, 2009; Pinós et al, 2009). These additional first exon sequences are all located upstream of the PL promoter, indicating that the SHBG gene utilizes at least six different promoters. We characterized transcripts derived from two of these upstream promoters, and found that they, too, undergo alternative splicing of exons 4-7. In total, from PL and these two upstream promoters alone, we identified 19 different SHBG gene transcripts (Nakhla et al, 2009); Pinós et al. describe additional transcripts arising from other SHBG gene promoters (Pinós et al, 2009). However, apart from the singular transcript encoding SHBG itself, it is unclear whether any other SHBG gene transcript encodes a functional protein in humans, or whether they might act to regulate expression of the SHBG transcript.
