Activation of the Androgen Receptor by Intratumoral Bioconversion of Androstanediol to Dihydrotestosterone in Prostate Cancer

The androgen receptor (AR) mediates the growth of benign and malignant prostate in response to dihydrotestosterone (DHT). In patients undergoing androgen deprivation therapy for prostate cancer, AR drives prostate cancer growth despite low circulating levels of testicular androgen and normal levels of adrenal androgen. In this report we demonstrate the extent of AR transactivation in the presence of 5α-androstane-3α,17β-diol (androstanediol) in prostate-derived cell lines parallels the bioconversion of androstanediol to DHT. AR transactivation in the presence of androstanediol in prostate cancer cell lines correlated mainly with mRNA and protein levels of 17β-hydroxysteroid dehydrogenase 6 (17β-HSD6), one of several enzymes required for the interconversion of androstanediol to DHT and the inactive metabolite, androsterone. Levels of retinol dehydrogenase 5, and dehydrogenase/reductase short-chain dehydrogenase/reductase family member 9, which also convert androstanediol to DHT, were lower than 17β-HSD6 in prostate-derived cell lines, and higher in the castration-recurrent human prostate cancer xenograft. Measurements of tissue androstanediol using mass spectrometry demonstrated androstanediol metabolism to DHT and androsterone. Administration of androstanediol dipropionate to castration-recurrent CWR22R tumor bearing athymic castrated male mice produced a 28-fold increase in intratumoral DHT levels. AR transactivation in prostate cancer cells in the presence of androstanediol resulted from the cell-specific conversion of androstanediol to DHT, and androstanediol increased LAPC-4 cell growth. The ability to convert androstanediol to DHT provides a mechanism for optimal utilization of androgen precursors and catabolites for DHT synthesis

A Regulatory Network for Coordinated Flower Maturation

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs

A Regulatory Network for Coordinated Flower Maturation

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs

Global analyses of gene expression in <i>arf6-2 arf8-3</i> and <i>myb21-5 myb24-5</i> stage 12 flowers.

<p>Venn diagram indicates numbers of genes with higher or lower expression in mutant compared to wild-type flowers, based on a t-test (P<0.05) and a two-fold ratio of expression values. Pie charts indicate the proportion of genes in each expression class having highest expression in sepals, petals, stamens, or carpels of wild-type stage 12 flowers <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002506#pgen.1002506-Schmid1" target="_blank">[21]</a>. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002506#pgen.1002506.s010" target="_blank">Table S3</a> lists these genes and provides details of their expression levels.</p

Inflorescence apices and flower phenotypes of <i>myb21</i>, <i>myb24</i>, <i>myb108</i>, and <i>aos</i> mutants.

<p>(A–H) Photographs of inflorescences (left panels) and individual flowers (right panels) of indicated genotypes. Asterisks indicate the position of the first open flower (stage 13) in the inflorescences shown, or the corresponding flower based upon bud size and position compared to a wild-type inflorescence. Individual flowers shown in the right panels are the first open flower (stage 13, A–F) or the fourth open flower (stage 15, G–H). Some sepals and petals have been removed to show inner organs. Scale bar: left panels, 3 mm, right panels, 1 mm. (I, J) Scatter plots showing petal and stamen lengths relative to gynoecium length of individual flowers of indicated genotypes. In I, data from a single experiment are shown. In J, measurements from two experiments were combined. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002506#pgen.1002506.s002" target="_blank">Figure S2</a> shows similar data for additional genotypes.</p

Gene expression and jasmonate production in wild-type and mutant flowers.

<p>(A–B) RNA gel blot hybridization using <i>MYB21</i>, <i>MYB24</i>, <i>MYB57</i> and <i>MYB108</i> probes. (A) RNA from wild-type, <i>arf6-2 arf8-3/ARF8</i> and <i>arf6-2 arf8-3</i> inflorescences (left panel), and wild-type stage 1–10, stage 11–12 and stage 13–14 flowers (right panel). (B) RNA from untreated (left panel) or MeJA treated (right panel) wild-type, <i>arf6-2 arf8-3</i>, <i>aos-2</i> and <i>coi1-1</i> inflorescences. (C) RNA gel blot hybridization using <i>ARF6</i>, <i>TPS11</i>, <i>TPS21</i>, <i>MYB108</i> and <i>SAUR63</i> probes. RNA from wild-type, <i>arf6-2 arf8-3</i> and <i>myb21-5 myb24-5</i> inflorescences. (D) RNA gel blot hybridization using <i>SAUR63</i>, <i>IAA2</i>, <i>IAA3</i>, <i>IAA4</i>, <i>IAA7</i>, <i>IAA13</i>, <i>IAA16</i> and <i>IAA19</i> probes. RNA from wild-type, <i>arf6-2 arf8-3</i> and <i>myb21-5 myb24-5</i> stage 12-13 flowers. (E) RNA gel blot hybridization using <i>LOX2</i>, <i>DAD1</i>, and <i>AOS</i> probes. PolyA⁺ RNA from wild-type, <i>arf6-2 arf8-3</i> and <i>myb21-5 myb24-5</i> stage 12–13 flowers. In A–E, numbers beneath each band indicate measured signal level relative to the <i>β-TUBULIN</i> control. (F) cis-JA concentrations in wild-type, <i>arf6-2 arf8-3</i>, <i>myb21-5 myb24-5</i>, and <i>aos-2</i> stage 12-13 flowers. Data are means of two measurements ± SD. n.d., not detected.</p

Phenotypes related to insect attraction.

<p>(A–D) Base of gynoecia of indicated genotypes. Arrows indicate nectaries. Scale bar, 0.1 mm. (E) Comparative quantitative analyses of floral volatile sesquiterpene emissions from wild-type, <i>myb21-5</i>, <i>myb24-5</i>, and <i>myb21-5 myb24-5</i> mutants. Emitted compounds were collected for 7 h from 40 detached inflorescences by a closed-loop stripping procedure. Emission was determined in ng h⁻¹ per 40 inflorescences. Values are averages and standard deviations of three independent collections. Only emissions of (<i>E</i>)-β-caryophyllene, the product of TPS21, and thujopsene, the product of TPS11, are shown. Different letters indicate significant differences in emissions of each compound between genotypes ( p≤0.001). (F) GC-MS analyses of sesquiterpene hydrocarbons collected via SPME from 20 inflorescences of wild-type, <i>myb21-5</i> and <i>arf6-2 arf8-3</i> mutants. Peaks marked with circles represent sesquiterpenes produced by the terpene synthase TPS21. Compounds not labeled with circles are products of the terpene synthase TPS11, with the exception of α-farnesene (α-farn). 1, (<i>E</i>)-β-caryophyllene; 2, thujopsene; 3, α-humulene; 4, β-chamigrene. Peaks marked with asterisks are other sesquiterpene products of TPS11 or TPS21 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002506#pgen.1002506-Tholl1" target="_blank">[43]</a>.</p

Genetic model of Arabidopsis flower maturation.

<p>(A) Diagram of principal regulatory pathways. Arrows indicate regulatory events established in this work or by previous studies. Both gibberellins and ARF6 and ARF8 auxin response factors promote jasmonate biosynthesis at flower stage 12. Auxin presumably enables ARF activity, and this may also be regulated by the circadian rhythm. Jasmonates in turn activate expression of genes for jasmonate biosynthesis, in a positive feedback loop requiring the JA-Ile receptor COI1. The underexpression of potential direct ARF6- and ARF8-targets in <i>myb21 myb24</i> flowers suggests that MYB21 and MYB24 may also participate in an additional positive feedback loop that promotes ARF6 and ARF8 activity, possibly through effects on auxin level (shown as dashed arrows). MYB21 represses jasmonate biosynthesis, and after the flower has opened (stage 13 and later), this negative feedback arrests flower maturation functions. In the absence of jasmonate signaling, ARF6 and ARF8 also contribute to <i>MYB21</i> expression in late-stage petals. (B) Illustration of flower developmental events regulated by the network between flower stage 12 (left) and stage 13 (right). The network induces downstream effectors that promote multiple events including petal and stamen filament elongation (regulated by ARF16 and by SAUR proteins), anther dehiscence (regulated by MYB108), volatile compound production (by TPS11 and TPS21 terpene synthases), nectary growth and development (regulated by CRC), and gynoecium growth and maturation. These and other effector genes may be activated directly or indirectly by MYB21 and MYB24, or by ARF6 and ARF8 independently of the MYB proteins. N, nectary.</p

Expression of <i>MYB21</i>, <i>MYB24</i>, and jasmonate pathway genes in wild-type and mutant flowers at stages 12, 13, and 14.

<p>Gene expression was measured by quantitative RT-PCR. Shown are means of two biological replicates each having three technical replicates (± SD). Within each biological replicate, expression levels were normalized to expression in wild-type stage 12 flowers.</p