Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains/losses, including ETS gene fusions, PTEN loss and androgen receptor (AR) amplification, that drive prostate cancer development and progression to lethal, metastatic castrate resistant prostate cancer (CRPC)1. As less is known about the role of mutations2–4, here we sequenced the exomes of 50 lethal, heavily-pretreated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment naïve, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPC (2.00/Mb) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1, which define a subtype of ETS fusionnegative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in ~1/3 of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Further, we identified recurrent mutations in multiple chromatin/histone modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with AR, which is required for AR-mediated signaling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signaling and increases tumour growth. Proteins that physically interact with AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX, and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signaling deregulated in prostate cancer, and prioritize candidates for future study.

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The second messenger cAMP has been implicated in the regulation of mammalian and amphibian oocyte maturation. Although a decrease in intraoocyte levels of cAMP precedes germinal vesicle breakdown (GVBD), the gonadotropin induction of ovulation and oocyte maturation is associated with major increases of cAMP in ovarian follicles. In the mammalian system, isolated oocytes undergo spontaneous maturation in vitro but this process is blocked by treatment with a phosphodiesterase (PDE) inhibitor, IBMX, which increases intraoocyte cAMP levels. In contrast, the same inhibitor, when added to cultured follicles for a brief time, increases follicle cAMP levels, followed by the induction of GVBD. To resolve the paradoxical actions of this PDE inhibitor on the maturation of isolated and follicle-enclosed oocytes, we hypothesized that meiotic maturation requires opposing fluctuations of cAMP levels in the somatic granulosa and germ cells. Such opposing fluctuations may result from selective expression and regulation of PDEs in the somatic and germ cell compartments of the follicle. To test this hypothesis, PDE activity was manipulated in different follicular cells using type-specific inhibitors. The impact of the ensuing changes in cAMP levels in the two compartments was monitored by the induction of GVBD. In isolated oocytes, spontaneous GVBD was blocked by two inhibitors of type 3 PDE (cGMP-inhibited: CGI-PDE), milrinone and cilostamide. In contrast, treatment with an inhibitor for type 4 PDE (cAMP-specific), rolipram, was ineffective. These findings suggest that the oocyte expresses type 3 but not type 4 PDE and that increases in intraoocyte cAMP suppress GVBD. This hypothesis was confirmed by in situ hybridization studies with PDE3 and PDE4 probes. PDE3B mRNA was concentrated in oocytes while PDE4D was mainly expressed in granulosa cells. In cultured follicles, LH treatment induced oocyte maturation but the gonadotropin action was blocked by inhibitors of type 3 but not the type 4 PDE inhibitors. Furthermore, treatment with the type 4, but not the type 3, PDE inhibitor mimics the action of LH and induces oocyte maturation, presumably by increasing cAMP levels in granulosa cells. Our findings indicate that PDE subtypes 4 and 3 are located in follicle somatic and germ cells, respectively. Preferential inhibition of PDE 3 in the oocyte may lead to a delay in oocyte maturation without affecting the cAMP-induced ovulatory process in the somatic cells. Conversely, selective suppression of granulosa cell cAMP-PDE may enhance the gonadotropin induction of ovulation and oocyte maturation. Thus, in addition to the well-recognized differential expression and regulation of adenylate cyclase in the somatic and germ cell compartments of the follicle, we suggest that selective regulation and expression of PDEs may be involved in the regulation of cAMP levels and control of oocyte maturation in the preovulatory mammalian follicle.

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