Prostate cancer co-opts a unique set of cellular pathways in its initiation and progression. The heterogeneity of prostate cancers is evident at earlier stages, and has led to rigorous efforts to stratify the localized prostate cancers, so that progression to advanced stages could be predicted based upon salient features of the early disease. The deregulated androgen receptor signaling is undeniably most important in the progression of the majority of prostate tumors. It is perhaps because of the primacy of the androgen receptor governed transcriptional program in prostate epithelium cells that once this program is corrupted, the consequences of the ensuing changes in activity are pleotropic and could contribute to malignancy in multiple ways. Following localized surgical and radiation therapies, 20-40% of patients will relapse and progress, and will be treated with androgen deprivation therapies. The successful development of the new agents that inhibit androgen signaling has changed the progression free survival in hormone resistant disease, but this has not changed the almost ubiquitous development of truly resistant phenotypes in advanced prostate cancer. This review summarizes the current understanding of the molecular pathways involved in localized and metastatic prostate cancer, with an emphasis on the clinical implications of the new knowledge. Prostate cancer (PCa) is a complex multifaceted and biologically heterogeneous disease. The majority of men diagnosed with prostate cancer will benefit from not being treated, because they have low volume indolent tumors that do not require immediate treatment. Overtreatment of localized PCA has become a serious problem, not in the least because of serious health risks involved in prostatectomy and other commonly used approaches. Moreover, biochemical relapse occurs in about 30% of patients who were treated aggressively.To minimize overtreatment of patients with indolent PCa, active surveillance is a reasonable and widely accepted approach  for manyAt the other end of the spectrum in localized PCa is the category of men presenting with either a high risk localized cancer or with metastatic disease. These are usually treated aggressively with any of the following: prostatectomy, radiation therapy and/or androgen deprivation therapies (ADT), which have been expanded in recent years to include novel substantially more efficient drugs. Nevertheless, even with newest ADT drugs, the outcome involves nearly inevitable progression to castrate resistant disease (CRPC), metastases and death. Moreover, a meta-analysis of primary ADT treatment provided conclusive evidence on the lack of survival benefit from PADT for most men with clinically localized prostate cancer .A relatively low rate of mutations in PCa compared to other tumors.Prevalence of non-random copy number variations (CNV) in most PCa tumors involving well-known prostate oncogenes or tumor suppressors.Recurrent chromosomal rearrangements involving ETS transcription factors, most frequently ERG, in 60 to 70% of PCa, which place these proteins under controls of an androgen-dependent promoter.Complex nature of the genomic rearrangements observed in PCa, with a pattern of balanced breaking and rejoining (“close chain” pattern). The highly rearranged PCa genomes are thought to evolve in a punctuated manner, with translocations and deletions occurring interdependently, via “chromoplexy” .Heavy involvement of developmental pathways that govern prostate embryonic development in the initiation and particularly progression to CRPC.The key role in prostate cancer of the epigenetic changes such as chromatin remodeling, DNA methylation and histone acetylation.The whole scale alterations in transcriptional programs, in particular those governed by androgen receptor (AR), and their prominent role in driving DNA rearrangements and co-opting developmental pathways.Continuum of genetic somatic changes in PCa from PIN  to CRPC (castrate resistant prostate cancer), i.e. increased frequency of changes already existing in primary PCa as disease progresses to CR stage, a well as development of new somatic aberrations in CRPC.The genetic landscape of prostate cancer was intensely explored in the last few years with NGS, whole genome expression analyses and analyses of epigenetic alterations. These findings, along with the results from genetically engineered mouse models (GEMM) for PCa initiation and progression revealed a number of features not encountered in their entirety in other cancers. These are:The most common known genomic alterations in PCa involve four pathways/genes: the androgen receptor pathway, PI3K pathway, rearrangements that place members of the ETS transcription factor family under control of androgen responsive promoter TMPRSS2, and loss of function of the prostate tumor suppressor NKX3.1. The other somatic alterations and pathways involved in PCa are listed in Table Risk stratification of localized PCa is a high priority, with an overarching goal of identifying groups of patients who will benefit from aggressive treatment approaches versus those whose disease will remain indolent for years and who are good candidates for active surveillance or even no intervention at all. Ideally, it should perform better than the current histopathological/clinical grading . However, the identification of molecular subtypes that drive differential prognoses in localized PCa was and remains a challenge.Localized PC could be (relatively but not entirely arbitrary) subdivided in two categories based on presence/absence of TMPRSS2-ERG or other changes in ETS family genes Figure . ETS famTMPRSS2-ERG fusion  is a resPatients with expression of ERG in high-grade prostatic intraepithelial neoplasia are more likely to develop prostate cancer. . ExpressBecause ETS transcription factors in fusion-positive tumors are expressed from an androgen-dependent promoter, their levels are significantly higher in these tumors. This is, no doubt, related to the biological role of ERG and other ETS in PCa. However, TMPRSS2-ERG expression was shown to persist in castration resistant prostate epithelial subpopulations which indicates that its expression might not be driven by androgen exclusively . PresencTMPRSS2-ERG expression induces repressive epigenetic programs by upregulating expression of the EZH2, a Polycomb group protein . OverexpDeregulation of WNT and TGFβ signaling pathways was also found to be associated with TMPRSS2-ERG fusion . As an oOther members of ETS family were also implicated in PCa via chromosomal rearrangements. ETV1 activation in a mouse models appears to have consequences for AR transcription that are different from those induced by translocated ERG: ETV1 largely cooperates with the AR transcriptional program, and promotes autonomous testosterone production . ETV1-poin vitro and in vivo by promoting degradation of ERG protein [Currently there are no drugs targeting ETS family transcription factors. In preclinical studies a compound WP1130, inhibitor of deubiquinating enzyme USP9X was shown to restrain growth of prostate cancer  protein .It was suggested that ETS fusion positive PCa patients could benefit from treatment with poly (ADP-ribose) polymerase 1 (PARP1) inhibitors because TMPRSS2:ERG interacts in a DNA-independent manner with PARP-1 and the catalytic subunit of DNA protein kinase (DNA-PKcs). Moreover, these interactions are essential for the transcriptional program of ETS factors . A randoActivation of phosphoinositide-3-kinase (PI3K) pathway, most often through PTEN copy losses occurs in 50% of PCa, and appears to be an early change, found already in PIN. PTEN is a phosphatase that is a well known as a tumor suppressor downregulating the PI3K pathway activity. PTEN deletions and/or mutations are found in 30% of primary prostate cancers  and 63% Other components of the PI3K pathway are also infrequently altered in PCa, such as mutations in PIK3 itself, in phosphatases other than PTEN - INPP4B and PHLPP , or in PStudies in GEMM strongly confirmed the role of PTEN in prostate carcinogenesis. The monoallelic ablation of PTEN in prostates of adult mice is sufficient to induce PIN that do not, however, progress to cancer . These mGenetic changes leading to activation of PI3K pathway through various mechanisms  are enriched in tumors positive for ETS fusions. Well-supported evidence exists, mostly from GEMM, of cooperation between ETS aberrations and PIK3CA pathway in development of PCa (see above). Not much information is available about the accompanying driver mutations in a relatively small subset of T/E positive tumors with normal PI3K/PTEN status.Aberrations of PI3K pathway contribute to development of the castration-resistance in PCa, at least in GEMM. Castration-resistant growth is an intrinsic property of Pten null prostate cancer cells, independent of cancer development stage . DeletioIn humans, numerous studies demonstrated the association between PTEN loss and worse prognosis, including shortened PFS  in partiCastration or treatment with Enzalutamide (AR antagonist) in a GEMM of high grade (HG) PIN that develops in absence of PTEN resulted in rapid progression of the otherwise stable HG-PIN to CRPC . HoweverMore and more evidence suggest that ADT benefits are reduced in PCa with PI3K activation , 49, andA recent report on failure of mTOR inhibitor temsirolimus in mCRPC patients suggests that a single targeted therapy is not sufficient to have an impact on the course of this disease . The facUntil recently, the driver mutations in ETS fusion-negative PCa were unknown. In the last year or two, a number of genomic aberrations that occur selectively in ETS fusion-negative PCa were identified, mostly through use of NGS and analysis of epigenetic alteration.SPOP mutations (6-15% of PCa) appear to represent a genetic subclass of PCa of its own. Mutations in SPOP are mutually exclusive with the ETS family rearrangements and rarely have accompanying mutations in PTEN or PIK3CA or TP53 in localized cancers. SPOP mutations define a subgroup of PCa with poor prognosis . They arin vitro prevents formation of ERG rearrangements due to impairment of androgen receptor (AR)-dependent transcription, a prerequisite for ERG translocation, which explains the mutual exclusivity of ERG rearrangements and CHD1 loss [CHD1. Loss of this chromatin remodeler occurs in 5-10 % of PCa, exclusively in ETS fusion negative tumors, and is frequently associated with mutations of SPOP , 60. CHDHD1 loss .SPINK1 overexpression, found in 5-10% of PCa is mutually exclusive with ERG rearrangements  and stroMethylation of miR-26a. ETS fusion negative PCa frequently are hypermethylated at the miR-26a locus . SystemaMAP3K7/TAK1. Deletion mapping of locus 6q12-22, one of the most commonly deleted loci in PCa has narrowed it to 6q15 and identified MAP3K7 as one of five genes present within it . TAK1 waThe role of AR signaling in the initiation of PCa remains to be fully understood. It might depend on the nature of the initiating oncogenic signal. As an example, ablation of AR in GEMM prevents development of PIN by FGF10 signaling (paracrine), but ablation of AR in GEMM does not prevent induction of PIN by activated Akt .Even though AR itself is never altered in primary PCa, about half of localized tumors harbor alterations in several of AR transcriptional cofactors/regulators . Among tNKX3.1 is frequently mutated or lost in localized PCa. The current understanding of the consequences of the loss of function of this tumor suppressor will be discussed in the section on metastatic CRPC, because frequency of NKX3.1 inactivation is much higher in advanced tumors, and because it is a gene essential in developmental processes that are discussed separately below.Classical tumor suppressors: inactivation of TP53, CDKN1B (p27/KIP), RB1 occurs infrequently in primary PCa, but is much more common in CRPC.MED12 is mutated in 5% of prostate cancer . It is aMYC overexpression is observed in PIN  and in pCADM2 is nectin-like member of the immunoglobulin-like cell adhesion molecules with expression reduced in PCa  and disrCRPC is characterized by massive accumulation of genomic and epigenetic alterations involving a number of developmental, signal transduction pathways as well as oncogenes and tumor suppressor controlled pathways Table . These aAR pathway is a driving force in CRPC, as seen from its deregulation in vast majority of these cancers. As described above, a significant number of localized cancers have perturbations in AR associated regulators and co-factors, but not in AR . HoweverPotential mechanisms by which AR reactivation occurs in CRPC include variable levels of AR gene amplification (30% of cases or higher), activating AR mutations, activating alternative mRNA splicing (10-25%), increased expression or activation of AR transcriptional coactivators, increased intratumoral androgen synthesis, activation of modulatory kinase pathways and noncoding RNAs (see below). All these alterations lead to sustained androgen receptor signaling in presence of castrate serum levels of androgen. The array of different mechanisms that contribute to activation of AR in CRPC is extremely diverse.Amplification of AR  occurs iMore recently, a mutagenesis screen identified a mutation F876L in AR that could convert the second generation AR antagonist enzalutamide into an agonist. This works also identified compounds that could antagonize AR F876L . F876L mAnother common mode of AR activation involves alternative splicing in AR , 95, leaGenomic rearrangements within the AR locus were discovered that prevent expression of full-length receptor but produce truncated versions lacking the androgen binding domain. These truncated proteins maintain the AR transcriptional program constitutively and in a truly androgen independent manner .Posttranslational modifications of AR. Multiple modifications of AR by phosphorylation, sumoylation, methylation and acetylation have been reported in the literature (reviewed in ), many oSomatic genetic changes in components of AR transcriptional co-regulators leading to an increased and/or changed output of AR activity. Mutational inactivation of inhibitory factors NCOR1, NCOR1 and NRIP1, and activating changes in NCOA1, NCOA2 and TNK2 are observed in primary cancers but are much more frequent in metastatic . AR acceIntratumoral androgen synthesis is increased through elevated endogenous expression of enzymes in the androgen synthesis pathways in tumors  or conversion of circulating low affinity adrenal androgens to DHT -107. AndUpregulation of AR signaling through activation of modulatory kinase pathways and AR phosphorylation. The cooperation of activated PI3K pathway in AR signaling was mentioned above in the section describing PTEN deletions. Signaling by activated Akt (as a results of PTEN loss) and ERK promote hormone-independent but AR dependent growth of PCa cells and tumors . In addiRegulation of AR degradation. Numerous publications describe multiple mechanisms of maintaining the stability of AR in CRPC. E3 ubiquitin ligases Mdm2  and CHIPAR is the primary treatment target in PCa. The development of novel therapies to achieve androgen deprivation in prostate cancer patients has improved the outlook for patients with advanced-stage and castration-resistant prostate cancer. However, in majority of patients the beneficial effects are self-limited, though some patients derive a long term or even life long benefit.In the recent decade or so, it was realized that some CRPCs remain hormone-dependent in spite of the very low levels of circulating androgens, due to some of the mechanisms described above. That led to the development and FDA approval of the new generation of drugs such as Abiraterone (inhibitor of enzyme CYP17 in the androgen synthesis pathway) and enzalutamide (selective AR inhibitor), with more in development Table . AbirateTOK-001, another CYP17 inhibitor in development, not only inhibits CYP17, but also target the AR receptor itself to prevent binding of androgens or even induce AR degradation . OrteronEnzalutamide has shown efficacy in CRPC patients whose disease progressed after chemotherapy  and in cNevertheless, it is becoming apparent that ADT can activate bypass pathways that can replace AR activity in presence of AR blockade and promote anti-androgen resistance. The recent demonstration that glucocorticoid receptor (GR) is upregulated and activated in PCa models involving continuous treatment with enzalutamide and ARN-509 is a striking illustration of the adaptability of PCa to ADT . MoreoveThere is an opinion shared by a number of researchers that many prostate cancers, in particular those with deregulated signaling pathways such as PI3K, should be treated with investigational therapies that target not only AR but these signaling pathways as well  or with Clinical trials are ongoing that target growth factor receptors, some in combination with ADT. Phase I/II NCT00953576 explores combination of lapatinib, small molecule inhibitor of EGFR and HER2 with dutasteride, inhibitor of 5-a-reductase. Multi-RTK inhibitor sunitinib and SRC family inhibitor dasatinib are being evaluated in a randomized trial NCT01254864 with abiraterone. Dasatinib versus placebo with abiraterone is in an additional phase II trial NCT01685125. MTD of dasatinib will be given to patients undergoing ADT (abiraterone) and radiation therapy in phase I trial NCT01826838 with the hope that inhibition of SRC pathway might overcome radioresistance.IGF1R is targeted with a humanized monoclonal antibody cixutumumab/IMC-A12. ADT (different drugs) with or without cixutumumab is tested in randomized phase II NCT01120236 for patients with newly diagnosed mCRPC. Cixutumumab is combined with mTOR inhibitor temsirolimus in phase I/II trial NCT01026623 for mCRPC.Cabozantinib/XL184 is a multi-RTK inhibitor with activity toward MET, VEGFR2 and other RTKs. The rationale of using it in prostate and other cancers is that it could potentially inhibit the angiogenic signaling in endothelial cells and the oncogenic MET signaling in tumor cells. Recent evidence shows that cabozantinib also restrains the activity of osteoblasts therefore inhibiting growth of bone metastases in mouse models . CabozanOther RTK inhibitors in clinical studies for CRPC include PLX3397 , antiangiogenic axitinib and pazopanib (VEGFR and PDGFR) and dovitinib (FGFRs and other RTKs).Introduction of abiraterone and enzalutamide into clinical practice gave new options to CRPC patients who had none before, but development of resistance ultimately limits the impact of these agents. A recent review described some of the clinical approaches to forestall or overcome resistance to new ADT agents , and intin vivo [https://www.endocrine.org/).One approach relies on blocking interactions of AR with its co-activators, which are essential for the activation of the AR transcriptional program. A peptidomimetic compound was designed that selectively targets protein motif LXXLL critical for interaction of AR with co-factors such as PELP-1, and showed a promising preclinical activity . A compoin vivo  and maybin vivo . A recenNiclosamide, another anthelmintic drug approved by FDA, was identified as a potent inhibitor of variant alternatively spliced AR (AR-V7) that drives resistance to enzalutamide in prostate cancer cells .in vitro and inhibited growth of CRPC in mouse models in vivo, presenting a new epigenetic approach [The recent demonstration of preclinical efficacy of inhibiting bromodomain and extraterminal (BET) proteins in different malignancies may be applicable in CRPC. BET domain protein BRD4 was shown to interact with the N-terminal domain of AR, and the BET domain inhibitor JQ1 disrupts AR transcription program approach .Targeting epigenetics turned out to be key to the activity of a compound identified initially as a active in a screen for drugs inhibiting translocations in prostate cancer. SD70 inhibits the androgen-dependent AR program, and prostate cancer cell growth, acting, at least in part, by functionally inhibiting the Jumonji domain-containing demethylase, KDM4C .in vitro and in vivo [Based on the observation that enzalutamide resistant PCa cells exhibit increased autophagy, a study of autophagy inhibitors found that CRPC cells are sensitive to their cytotoxic action  in vivo .The role of the PI3K pathway in the development of PCa and CRPC, and the reciprocal feedback regulation of PI3K and AR activities in particular gained even more importance in light of recent findings. Loss of PTEN in PCa is apparently strongly co-operative with other somatogenic changes in the development of the CRPC phenotype. A co-clinical study of GEMM with PTEN loss in prostate revealed that resistance to ADT on this background develops only in presence of additional alterations – in this scenario, loss of ZBTB7A or p53 . This stThe second study has found that KLK4  and PLZF (promyelocytic leukemia zinc finger), two genes upregulated by AR, contribute to integration of AR and mTOR signaling. KLK4, long suspected as a player in PCa, apparently destabilizes PLZF through direct interaction and therefore abrogates the negative effects of PLZF on AR transcriptional activity . MoreoveActive clinical research is being undertaken to examine how inhibition of signaling pathways initiated by activated receptor kinase and mediated through the PI3K pathway might affect the course of CRPC.Of more than 20 experimental drugs with activity against PIK3 kinase, three, BKM120, BEZ235 and GDC-0980, are currently tested in several phase II clinical trials selectively for PCa. Phase II trial NCT01385293 is recruiting patients with mCRPC for a single arm study of BKM120 at a pre-determined maximum tolerated dose. Phase Ib NCT01634061 will examine combination of either BKM120 or BEZ235  with abiraterone in patients with CRPC. Similarly, combination of BKM120 and abiraterone will be tested in NCT01634061. BEZ235 is in another multicenter trial with abiraterone, NCT01717898. Dual PI3K/mTOR inhibitor GDC-0980 is tested in a randomized phase II NCT01485861 with abiraterone. Several other PI3K inhibitors are in early clinical testing  for various cancers, including prostate.AKT inhibitor GDC-0068 is tested in a randomized phase II trial NCT01485861 with abiraterone. The phase II randomized trial NCT01251861 testing bicalutamide alone or bicalutamide with AKT inhibitor MK2206 in patients for previously treated PCa. AKT inhibitor AZD5363 is in phase I testing, NCT01692262.mTOR inhibitors everolimus and temsirolimus, approved for other conditions, are in early clinical testing in PCa. Combinations of temsirolimus with docetaxel (NCT01206036) and with vorinostat (NCT01174199) are in phase I testing. Everolimus with radiation treatment is explored for biochemical recurrence after prostatectomy NCT01548807, and as an add-on for patients undergoing radiation treatment with ADT NCT01642732.Defects in DNA damage repair (DDR) in CRPC. Mutations in the well-known DDR genes have been reported in CRPC and in localized aggressive cancers. Mutations of BRCA2 were identified in about 2% of sporadic PCa, but germline mutations in BRCA2 increase risk of PCa at younger age (<55 years) manifold . Absolutin vitro and in vivo.As mentioned above, the DNA damage repair (DDR) protein PARP-1 is essential for the activity of TMPRSS2-EGR in PCa , but alsProstate tumors with mutated or deleted BRCA genes and ATM are candidates for treatment with PARP inhibitors in clinical trials. Several trials are ongoing, and at least two are testing PARP inhibitors in selected cancers (including PCa) with mutations in BRCA genes (phase I NCT00892736 with veliparib) and phase II NCT01078662 with olaparib.AR and DNA damage repair crosstalk. Importantly, results from large clinical trials showed strong augmentation of efficacy of radiotherapy (RT) for aggressive PCa when combined with anti-androgen therapy, suggesting a potential role for AR inhibition in dampening DDR. Two recent independent studies elucidated the role of AR signaling in enhancing DDR. AR promotes expression and activity of key DDR factors such as DNAPK, XRCC2, and XRCC3, whereas DNAPK in turn supports the AR transcriptional program . AndrogeMYB protein was found to supplant the role of AR in regulating DDR by regulating an overlapping set of genes. Knockdown of MYB or some of its targets  in CRPC increased the cytotoxicity of PARP inhibitor indicating that co-targeting MYB pathway and PARP activity could be a potential treatment strategy .Development of prostate is entirely dependent on endocrine and paracrine AR signaling, whereby expression of AR in UGS  mesenchyme orchestrates outgrowth and branching of prostatic epithelium, and the subsequent expression of AR in the epithelium is required for the production of prostatic secretion. It is now clear that developmental pathways activated by mesenchymal AR signaling and involved in the epithelial–mesenchymal interactions during prostate development could be inappropriately reactivated during tumorigenesis. These pathways are numerous  is mutated in 5% of prostate cancers , and mutin vivo . Severalin vivo . The latin vivo  and induin vivo . Two ageHedgehog pathway is an essential pathway in normal prostate embryogenesis . Sonic hThe role of Hh signaling in PCa is most likely associated with its ability to modulate activity of AR , 186. Hhin vitro for resistance to docetaxel are sensitive to the dual inhibition of Hh and Notch pathway by cyclopamine (inhibitor of Smo) and DBZ (inhibitor of γ-secretase).Hh and Notch pathways are involved in the development of resistance to docetaxel which is associated with elevated signaling and increased expression of Gli1 and Gli2 . Cells wAs described above, stabilization of GLI factors is one of the probable effects of SPOP mutations in PCa since SPOP participates in a pathway leading to the degradation of Gli . ActivatThe SMO-targeting agents GDC-0449 and LDE225 are in Phase I/II NCT01163084 trial and entering phase I NCT02111187, respectively, for locally advanced PCA; the non-specific Hedgehog pathway inhibitor itraconazole is in phase II trial NCT01787331 for patients with biochemical relapse and in combination with orteronel in phase I/II NCT02054793 for CRPC.TGF-β pathway and SMAD4. The role of TGF-β pathway in PCa, similar to other cancers, is complex. TGF-β is known to play a dual role in tumorigenesis, acting as a growth inhibitory tumor suppressor early in the process, and as a tumor promoter in late-stage disease. In a GEMM model of prostate tumorigenesis PTEN inactivation drives formation of indolent tumors and elicits the activation of TGF-β/BMP-SMAD4 signaling. The latter induces cellular senescence to curb tumor progression, and genetic deletion of SMAD4 (key effector in TGF-β pathway) leads to the development of highly invasive and metastatic tumors with 100% penetrance . This stThe TGF-β/SMAD4 dependent barrier to tumor progression is destructed in metastatic PCa through involvement of transcription factor COUP-TFII/NR2F2. COUP-TFII exerts its effects on TGF-β pathway by directly interacting with and inhibiting SMAD4, therefore cooperating with PTEN loss in GEMM . COUP-TFIn contrast to its barrier role during cancer initiation, TGFβ promotes metastatic phenotype in late stages by driving epithelial mesenchymal transition. TGFβ and TGFβR are expressed at higher levels in metastatic PCa, and are instrumental in EMT that is mediated, in part, by upregulation of the molecular chaperone clusterin via EMT transcription factor TWIST1 . While SRadiation therapy frequently employed for treatment of PCa can increase levels of serum TGFβ and promote distant metastasis. Clinical trial NCT01427322 aims to examine if the EGFR/HER2 inhibitor lapatinib given prior to palliative irradiation for bone metastases could lower the levels of TGFβ.Notch pathway. Notch signaling was shown to be critical for normal prostate development  using a Polycomb group protein EZH2. Enhancer of zeste homolog 2 is a methyltransferase and a component of repressive PRC2 complex that triggers transcriptional repression by catalyzing the addition of methyl groups onto lysine 27 of histone H3 (H3K27me2/3). EZH2 is not expressed in normal adult prostate, but is highly expressed in UGS during development and then again at puberty in prostatic epithelium . EZH2 exEZH2 could impede epithelial differentiation and contribute to prostate cancer progression because it was shown to directly modulate the transcriptional output of AR . Moreovein vitro against PCa cells [Inhibitors of EZH2 GSK126 and EPZ-6438 are in clinical trials for DLCBL and FL where EZH2 is frequently mutated on Y641 and A677. Another inhibitor, 3-deazaneplanocin A, has been reported to have activity Ca cells .Polycomb group protein BMI1 is a member of repressive and most likely oncogenic PRC1 complex acting in epigenetic silencing of gene expression. PRC1L monoubiquitinates nucleosomal histone H2A at lysine 119 . It stimBMI1 is induced by IKKa via transcription factor E2F1 in regenerating prostate and in PCA after ADT. This is a cell-autonomous process triggered by infiltrating B cells, and links CRPC development to inflammation . The regHOXB13 is a homeobox transcription factor that plays a critical role in prostate development. A variant HOXB133 G84E was found to be closely associated with the risk of prostate cancer . Hoxb-13FGFR pathway is intimately involved in the prostate development from urogenital sinus  through Inhibitors of FGFR signaling are in clinical trials. In particular, as FGFR was implicated in EMT and osteoblastic progression of PCa, a small molecule multikinase inhibitor dovitinib (TKI258) is explored in phase II trial for bone metastatic CRPC NCT00831792. Another inhibitor, nintedanib (BIBF1120) completed phase II trial NCT00706628, but results have not been reported.in vitro results in massive redistribution of AR binding sites, with some being highly enriched for AR binding, while other depleted of AR [in vitro are quite different from those identified in vivo, with AR-FOXA1 binding diminished and AR-STAT5 binding increased [FOXA1 is a transcription factor with a well-known essential role in prostate morphogenesis . It appeed of AR . The FOXed of AR . The prencreased . FOXA1 fncreased . Mutationcreased .Epigenetics encompasses several processes, such as DNA methylation, histone modifications and RNA interference. All of these are altered in PCa initiation and more so during progression, and play a functional role in prostate carcinogenesis. The role of epigenetic deregulation is PCa and particularly CRPC is strongly supported by a number of somatogenic alterations in multiple genes whose products are involved in DNA or histone modifications. These alterations result in whole-scale changes in DNA methylation and histone acetylation. Historically, hypermethylation of DNA has been long known to occur in PCa, with GSTP1 discovered as one of the prominent targets . RegionsIntercommunication of paracrine signaling and epigenetic alterations was demonstrated in a number of studies, where targeted inactivation of TGFβ receptor  or overeActivation of AR transcriptional program in CRPC apparently involves numerous chromatin remodeling events. For example, genomic studies have recently revealed that AR might act as a global transcriptional repressor. In embryonic stem cells, androgen-responsive elements (ARE) in AR-repressed genes are occupied by repressive Polycomb group protein EZH2 that maintains the undifferentiated state. These genes are also silenced in castration-resistant prostate cancer conferring to them a stem cell de-differentiated phenotype and promoting tumor progression . TranscrEZH2 and BMI1, already described above, are prime examples of how components of chromatin remodeling complexes are involved both in development of prostate and in PCa. CHD1, frequently inactivated in PCa , 61, is The chromatin-remodeling complex SWI/SNF plays a tumor suppressor role in PCa which is antagonized by a long noncoding RNA SChLAP1 . In partInactivation of tumor suppressors TP53 and RB1 is much more common in CRPC than in localized cancers -242. LosA recent study suggests that metastasis suppressor p63 inhibits EMT and metastases at least in part via regulation of miR-205. Either or both p63 and miR-205 are absent in lymph nodes or distant metastases of PCa patients .MYC amplification is very common in CRPC, found in at least a third of these tumors. As mentioned earlier, MYC is overexpressed already in PIN but amplifications are mostly limited to CRPC . C-Myc aRAF. Rearrangements of RAF oncogenes  are found in 1-2% of PCa, mostly CRPC . These rUBE2C is overexpressed in many tumors, including CRPC . UBE2C iEstrogen receptor β. Estrogens were originally used to treat PCa to reduce the hypothalamic pituitary stimulation of LH/FSH production and further reduce the synthesis of androgens in 1942, reprinted in . HoweverERβ agonist induces apoptosis in prostatic stromal, luminal and castrate-resistant basal epithelial cells in BPH of estrogen-deficient aromatase knock-out mice, as well as in xenografts of prostate cancer. ERβ is downregulated in high grade PCa via TGFβ and hypoxia, and loss of ERβ is sufficient to promote EMT in PCa. ERβ expression induces destabilization of HIF-1α and transcriptional repression of VEGF-A . The mecERβ agonist treatment attenuates clonogenicity and self-renewal of murine prostatic progenitor cells and depletes both murine and human prostatic basal cells. Subsequent to castration ERβ induces further apoptosis in basal, luminal and intermediate cells .ERβ ligands are not currently in clinical development for PCa, either as monotherapy or in combination with ADT.in vitro and give rise in vivo to basal, luminal and neuroendocrine cells thus exhibiting multipotency [in vivo, and luminal cells produce only luminal cells under these circumstances, with both lineages self-sustained in the normal adult prostate [Much controversy existed and some still exists in the field concerning the nature of prostate and PCa stem cells. The prostate gland contains epithelial luminal cells with high levels of expression of AR, basal cells with low or absent AR and rare neuroendocrine cells. There is an agreement regarding the origin of prostate stem cells, which have been amply demonstrated to reside in the basal multipotent population -266. A sipotency . Howeveripotency , 269. Thprostate .Lineage-tracing approaches have identified rare luminal cells, named castration-resistant Nkx3-1-expressing cells, or CARNs, as also possessing multipotent stem cell activity . HoweverIn terms of PCa initiation, evidence exists that supports involvement of both lineages. Prostate luminal cells, transit-amplifying cells , and basal cells have all been implicated as the cells of origin for prostate cancer. Basal cells with introduced relevant mutations demonstrated ability to form PCa thus supporting their initiating role in PCa. The tumor-initiating ability was found to reside in basal cells CD49fhiTrop2hi expressing p63 , 271.At the same time, the rare luminal cells, CARN, could also give rise to PCa on a PTEN null background . In lineThe cancer stem cells (CSC) of basal or liminal origin are very likely a source of treatment-resistant cells. The studies mentioned above described castration-resistant cells in both CSC populations. The prostate CSC do not express androgen receptor, or have very low levels of it, and therefore survive the androgen deprivation serving as a reservoir of treatment-resistant cells . These pTo support this notion, a cell population characterized by low levels of PSA (PSA(-/lo)) was identified as being quiescent, refractory to androgen deprivation, having high clonogenic potential and long-term tumor-propagating capacity. These express stem cell genes and can undergo asymmetric cell division to generate PSA(+) cells. PSA(-/lo) PCa cells resist androgen ablation in castrated hosts, and they harbor highly tumorigenic castration-resistant PCa cells. PSA(-/lo) cells may represent a critical source of castration-resistant PCa cells .in vivo [Apparently, some phenotypic markers of PCa stem cells might have a functional significance in development of PCa. Trop2hi has been shown to play a significant role in stem cell renewal and epithelial hyperplasia via β-catenin pathway. Trop2 undergoes intramembrane proteolysis to release two polypeptides, of which the intracellular one translocates to the nucleus. High expression of the Trop2 intracellular domain promotes self-renewal through signaling via β-catenin and is sufficient to initiate precursor lesions to prostate cancer in vivo .The role of the Polycomb group protein BMI in regeneration of normal prostate progenitor cells and in PCSC was described above. A recent publication elucidated a signaling axis involved in both normal prostate regeneration and in emergence of CRPC after ADT that consists of IKKa-E2F1-BMI1. Nuclear IKKa controls CRPC development through expansion of BMI1+ progenitors. The most intriguing aspect of these findings is that expression of BMI1 is triggered by inflammation that depends on the infiltration of B cells into regenerating prostate rudiments, either normal or cancerous, after ADT. The BMI1 controlled tumor growth is therefore at least partially cell-autonomous .Integrin β4 was shown recently to promote self renewal of putative cancer stem cells that are basal in origin. β4 promotes adhesion of the cells to the basal membrane, which apparently is necessary for the maintenance of stemness. More importantly, mutation of β4 prevents tumor formation on PTEN null background. Finally, the high level of expression of integrin β4 in prostate cancers was associated with androgen independent metastases to bone. Finally, integrin β4 is associated with activation of Erbb2 and Met receptor tyrosine kinases, and pharmacological inhibition of these results in efficient inhibition of tumor growth in mice . This inRecently, a report was published that described establishment of a xenograft model capable of supporting growth of stroma-supported xenografts from multiple patients with early stage disease . More imEMT endows cells with migratory and invasive properties, induces stem cell properties, and prevents apoptosis and senescence, thus orchestrating the initiation of metastasis. EMT is characterized by the loss of expression of E-cadherin and induction of N-cadherin, loss of cell polarity and dependence on adhesion, all contributing to metastatic phenotype. Numerous pathways have been implicated in EMT in PCa, including some developmental pathways, inflammation driven signaling, ERG fusions and others, some of which are listed below.in vitro and in patients. This transition was observed in normal prostate upon ADT and in PCa patients treated with ADT, and involves transcription factor ZEB1 [in vitro.Androgen deprivation induces expression of N-cadherin and EMT  in vitrotor ZEB1 . In additor ZEB1  and PDGFtor ZEB1  that proin vitro [EZH2 can induce EMT and increase the metastatic potential of prostate cancer cells by downregulation of DAB2IP, a tumor-suppressive Ras GTPase-activating protein (RasGAP) , 283. EZin vitro . SOX4 apin vitro .TMPRSS2/ERG was also shown to promote EMT via direct transcriptional activation of expression of ZEB1, and indirect activation of ZEB2 through IL1R2 and SPINT1 . In addiin vitro and in patients [in vitro [TGF-β represents a potent EMT inducer in normal development and tumor progression via Smad-dependent and independent transcriptional pathways . Smad-mepatients . TWIST1 in vitro , suggestin vitro and metastasis in vivo by disrupting the signaling leading to oxidative stress, hypoxia and EMT [Monoamine oxidase A (MAOA), a mitochondria-bound enzyme, was recently implicated in EMT in PCa. MAOA catalyzes the degradation of monoamine neurotransmitters and dietary amines producing peroxide as a by-product and increasing levels of cellular ROS. Expression of MAOA is associated with high grade PCa , and cau and EMT .Expression of inducible FGFR1 in a mouse model induces PCa with EMT characteristics and involves activation of SOX9 transcriptional activity and activation of WNT pathway protein Fzd4; this was validated in human PCa .in vitro [β2-microglobulin is a pleiotropic signaling molecule that is highly expressed in bone metastases in PCa. β2-M interacts, among many other proteins, with hemochromatosis protein HFE, modulating iron homeostasis and leading to activation of HIF-1 (hypoxia-inducible factor-1) signaling pathways . HIF1 acin vitro . Estrogein vitro .Paracrine interactions are also contributing to EMT in PCa. One mechanism involves tumor secreted IL-6 that elicits secretion of metalloproteases by stroma . Cancer Another example of the role that paracrine interactions play in EMT and metastasis of PCa involves tumor secreted cytokine CXCL16 and its receptor CXCR6 expressed on bone marrow derived mesenchymal stem cells (MSC). CXCR6 induced signaling on MSC promotes their recruitment into PCa tumors, conversion into cancer – associate fibroblasts (CAF) and secretion of CXCL12/stroma derived factor 1. In turn, CXCL12 stimulates PCa cells expressing the CXCL12 receptor, CXCR4, which facilitates EMT, migration and metastasis .EMT might play a critical role in the metastatic behavior of PCa, in particular bone metastases that present a very serious clinical challenge. Some investigational agents that might show efficacy in the spread of bone metastases are listed in Table NEPC is a subtype in a poorly defined group of prostate cancers that are variously described as “anaplastic”, “small cells PCa”, or simply “aggressive” and may represent different histopathological entities. These are associated with at least one of the following characteristics: exclusive visceral metastases, or predominantly lytic bone metastases, bulky tumors, low prostate-specific antigen, lack of or short response to androgen deprivation therapy and good but short-lived responses to platinum-based chemotherapy . ClassicIt has been suggested that introduction of the new ADT agents abiraterone and enzalutamide has significantly increased the emergence of castrate-resistant cancers with neuroendocrine features and visceral metastases -305. TheA first-in-class xenograft model established with xenografts from the fine-needle biopsies showed that neuroendocrine PCa can evolve directly from adenocarcinoma via an adaptive response after prolonged exposure to androgen withdrawal . PossiblMolecular changes strongly associated with NEPC were identified by NGS of RNA, and most frequent are overexpression of EZH2 and amplification of Aurora kinase A and NMYC . ConcurrThe neuroendocrine phenotype in PCa was shown to be mechanistically linked to the downregulation of transcriptional complex REST . REST isin vitro and in a mouse neuroblastoma model demonstrated that inhibition of AURKA with MLN8237 or MLN8054 actually triggers degradation of NMYC mediated by the Fbxw7 ubiquitin ligase [In addition to classic chemotherapy regimens, potential novel treatment options for this clinically aggressive type of PCa could involve Aurora kinase inhibitors. Clinical trials with inhibitor MLN8237 are ongoing (NCT01799278) or planned (NCT01848067), in combination with abiraterone or chemotherapy. A recent study performed n ligase .Search for diagnostic and prognostic tests in localized PCa. Testing for PSA prostate-specific antigen as a screening tool for PCa has been useful in diagnosis and follow-up to treatments in PCa, but it has shortcomings. These include false positives, unnecessary treatments for men with low grade PCa but elevated PSA, and occasional lack of PSA in high grade PCa (particularly with NE phenotype). Inter-individual variations in PSA levels have been reported to be associated with three particular polymorphisms in the individual genomes , and thiDetection of TMPRSS2-ERG (T/E) fusion in urine in combination with serum PSA was reported to be successful in risk stratification for PCa . A combiThe predictive value of testing is particularly high in active surveillance, a treatment (or lack of treatment) approach that has been supported by several recent clinical trials. They demonstrated better quality of life for low-risk prostate cancer patients who were actively monitored rather than treated for their disease. One such study, the Prostate Cancer Intervention Versus Observation Trial (PIVOT), found that men who have low-risk cancer may not need early treatment for prostate cancer . HoweverA recent study has reported identification of a 19 gene expression signature enriched in genes associated with aging and senescence, which allows to distinguish indolent low Gleason tumors. Moreover, expression of just three genes: FGFR1, PMP22, and CDKN1A accurately predicted outcome of low Gleason score tumors. Protein expression of this three-gene panel in biopsy samples distinguished Gleason 6 patients who failed surveillance over a 10-year period , but theConfrimMDx test from MDxHealth examines methylation status of a three genes, GSTP1, APC and/or RASSF1 in PCa biopsies. The company claims that their test is more accurate in detection of PCa in biopsies, because it could detect the effects of cancerous growth in cells adjacent to it, and does not have to rely on the identification of PCa foci in the needle core biopsies.Prognostic tests for CRPC. Significant work has been conducted in order to develop a minimally invasive diagnostic procedure, considering the risks associated with needle biopsies and the fact that biopsies of metastases present a number of risks and limitations. “Liquid” biopsies or predictive gene signatures based on DNA and mRNA analyses of whole blood are being developed. In one study, an expression signature of six genes was highly effective in predicting survival , and, siIsolation and analysis of circulating tumor cells (CTC) is a developing technology that is promising in metastatic cancers. According to several studies, the mere enumeration of CTCs in blood samples is prognostic and could be predictive of response to therapies in CRPC -332. CapReactivation of AR signaling despite continuous treatment with new drugs such as abiraterone (CYP17A1 inhibitor) is a common phenomenon. Prediction of response in patients is highly desirable, and a non-invasive test is much preferred to repeated biopsies. Isolation and analysis of CTC was explored as a diagnostic or prognostic factor for ADT. Earlier attempts to analysis of CTC were limited to quantification only. The feasibility of measuring the AR pathway activity in CTC was demonstrated . This apOther possibilities for novel non-invasive tests include mRNA seq in captured CTC  and deteThere has always been an interest in development of immunotherapeutics for PCa, and the only approved cell-based immunotherapy, Sipuleucel-T, was developed for PCa. Numerous other approaches are in clinical development Table , some ofA growing number of trials are ongoing with the immune checkpoint antibodies in prostate cancer. Ipilimumab, FDA approved anti-CTLA4 antibody, is in several trials in PCa, including randomized phase III NCT01057810 for patients with asymptomatic mCRPC and randomized phase III trial NCT00861614 with ipilimumab or placebo administered after radiotherapy. The results of latter trial failed to reach the primary endpoint of increasing OS, but showed some signs of activity of ipilimumab that warrant further investigation . The secIn spite of successes achieved with anti-PD-1 and PD-L1 antibodies in other malignancies, there are only two clinical trials with these agents ongoing for prostate cancer. NCT01420965 combines sipuleucel and anti-PD-1 antibody CT-011, and anti-PD-L1 antibody MSB0010718C is tested in a several cancer types including prostate. The relative dearth of trials with checkpoint antibodies is most likely due to the fact that in the trial of BMS-936558, anti PD-1 antibody, remarkable responses were observed in patients with melanoma, NSCLC and RCC, but not PCa . Immunosin vitro with CAR (chimeric antigen receptor) recognizing PSMA. Treatment will involve myeloablation with cyclophosphamide in patients with CRPC.Phase I trial NCT01140373 for mCRPC patients is testing harvested autologous T cells transduced An early phase clinical study is exploring potential of natural killer (NK) cells in various malignancies including PCa. In particular, this phase I trial (NCT00720785) will examine if the limited anti-tumor activity of NK cells could be significantly increased by pretreating patients with proteasome inhibitor bortezomib, which has been reported to enhance the sensitivity of tumor cells to NK killing in numerous studies , 341. InPROSTVAC-V and PROSTVAC-F are vaccinia and fowlpox based virus vaccines expressing PSA and TRICOM , and with GM-CSF. PROSTVAC-V is given for priming, and PROSTVAC-F for boosting the response. A phase II trial was completed without clear clinical benefits, such as increase in PFS, observed. However, the clinical evaluation of PROSTVAC-V/F should be re-considered considering different endpoints for immune therapies . Indeed,Phase II trial NCT01341652 examines PAP vaccine plus GM-CSF versus GM-CSF alone in non-metastatic PCa. Adenovirus/PSA vaccine is tested in Phase II NCT00583024 in hormone-refractory PCa, while NCT00583752 will test the same vaccine in men with locally treated PCa.in vitro with the recombinant fusion protein consisting of prostatic acid phosphatase (PAP) and GM-CSF. This causes the activation and expansion of the autologous antigen-presenting cells (APCs) and lymphocytes, even though the precise mechanism is still unknown. Treatment with Sipuleucel-T does not have an effect on levels of PSA or radiological parameters of disease, but has a modest effect on OS.Sipuleucel-T/Provenge/ was the first cellular immunotherapeutic to be approved by the FDA to treat cancer. This treatment consists of autologous peripheral blood mononuclear cells (PBMCs) enriched for a CD54+ DCs (dendritic cells). These are primed Sipuleucel-T is also examined as a neoadjuvant in patients with localized PCa: phase II, NCT00715104; in combination with external beam radiation therapy in CRPC patients , and with abiraterone in phase II NCT01487863. Combination of Sipuleucel-T with Ipilimumab, an immune checkpoint antibody targeting inhibitory CTLA-4, is in a phase II trial NCT01804465 which examines immediate versus delayed CTLA-4 blockade, and in phase I NCT01832870 for advanced PCa. A trial of Sipuleucel-T with another checkpoint antibody, CT-011 targeting PD-1 is also ongoing .BPX-201 DCs vaccine with activating agent AP1903 is undergoing testing in phase I trial NCT01823978 for mCRPC. DCs in this trial are transduced with adenovirus-based vector expressing PSMA and a fusion protein composed of synthetic inducible adjuvant iMC, drug-inducible costimulatory CD40 receptor (iCD40) and the adaptor protein MyD88, with potential immunomodulating and antineoplastic activities. The iCD40 contains a membrane-localized cytoplasmic CD40 domain fused to the FK506 modified drug-binding protein 12 (FKBP12). Upon intradermal administration of BPX-201, these DCs accumulate in local draining lymph nodes. Twenty-four hours after vaccination, the dimerizing agent AP1903 is administered. AP1903 binds to the drug binding domain, leading to iMC oligomerization and activation of iCD40 and MyD88-mediated signaling in iMC-expressing DCs.An exciting new target in treatment of PCA is neoneurogenesis, or the ingrowth of new nerve endings into a tumor. It was discovered recently that the autonomous nervous system plays a direct role in PCa growth and metastasis. Sympathetic and parasympathetic nerves in the normal prostate control the physiological function of both muscle fibers and epithelial compartment, but their involvement in PCa was unsuspected. As was demonstrated in , adrenerin vivo [A recent population study performed in Toronto showed that increasing duration of metformin use among diabetic men after a diagnosis of prostate cancer was associated with decreased prostate cancer–speciﬁc and all-cause mortality . The finin vivo . Metformin vivo .L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mTORC1 signaling. LAT1 and LAT3 are overexpressed in PCa, and they are responsible for increasing nutrients and stimulating cell growth. LAT3, in particular, is expressed at high levels in all stages of PCA, and its expression is suppressed after ADT . PharmacA recent study uncovered how two lncRNAs, PRNCR1 and PCGEM1, overexpressed in many CRPCs, concordantly enhance AR transcriptional activity in a complicated series of events. These lncRNAs localize to chromatin in androgen response area, whereby PRNCR1 binds the acetylated carboxyl end of AR, recruits methylase DOTL1, which methylates the amino terminus of AR, a pre-requisite to binding of PCGEM1. AR-bound PCGEM recruits protein Pygo2 bound to H3K4me3 on chromatin in the promoter area, thus inducing chromatin looping that brings enhancer and promoter area into close proximity. The proximity of AR bound enhancer and promoter sequences of target genes results in enhanced transcription of AR target, many of which contribute to oncogenesis . These lPresence of HHV-8  in normal prostate, prostate cancers and biological fluids was reported in a number of publications, some of which documented higher prevalence of HHV-8 in prostate cancer or seropositivity for HHV-8 in PCa patients, while others reported no such association. In general, HHV-8 infection was not linked etiologically to PCa. However, a recent publication reported that HHV-8 infection of androgen-responsive PCa cells confers androgen-independent growth via activation of EZH2 controlled gene silencing . These fEven though NGS studies were conducted for prostate cancer, the identification of well defined and clinically meaningful subtypes based on genomic profiling has been difficult. It is likely due to the high number of molecular alterations that contribute to the development of localized PCa. For example, about half of prostate cancers have translocations involving the ETS family members, but these are not sufficient to cause frank PCa. The additional alterations that cooperate with deregulated ETS are many, and presumably each of them  could determine the precise clinical subtype of the emerging tumor. The other known drivers of the PCa early development (in fusion-negative cancers) are also several: SPOP mutation, SPINK overexpression, CHD1 deletion and TAK1 loss, as well as chromosomal losses not characterized in terms of genes involved. These molecular subtypes of PCa await analysis of the clinical significance of the underlying somatic changes.The pervasive involvement of AR signaling in the later stages of PCa presents a conundrum as well, because it affects an entire massive transcriptional program that is further affected by additional alterations in tumor suppressors, transcription factors, chromatin remodeling enzymes and, almost universally, developmental factors. These multiple perturbations in advanced PCa present great difficulties in terms of not only treatments, but also identification of biomarkers of risk or prognostic/theranostic value.de novo alterations in other signaling, developmental or epigenetic pathways that bypass the need for androgen signaling.Since androgen regulated pathways are affected in the vast majority of advanced PCa, the ADT is a logical therapeutic intervention. The persistent significance of the AR signaling in CRPC was recently validated by the evidence of the clinical efficacy of androgen synthesis inhibitors (abiraterone) and the novel, second-generation AR antagonists . However, ADT is often used under circumstances where the mechanisms of resistance to it are most likely already present in a subpopulation of PCa cells. One possibility is the existence of a compartment of the androgen-independent cells with characteristics of stemness that already have the driver mutations such as ETS translocation or PTEN loss, and that are propelled into reproduction and acquisition of further genetic changes by the drop in androgen levels. Alternatively, a population of androgen independent or almost independent cells might emerge as a consequence of the ADT by selecting rare cells with The universal development of resistance to ADT, including the newest agents, is a testimony not only to the adaptability of the activity of AR pathway to the low or even absent androgens, but also an evidence of enormous adaptability of PCa cellular oncogenic pathways. Resistance to ADT is enormously important clinically, and is the subject of intense research (reviewed in , 353. EmIt is becoming a shared understanding that in the future, ADT will be administered as one component in a combination of therapies, to try and forestall the development of resistance. However, the real conundrum will be to find a right “partner” to the ADT. The problem lies in the identification of the particular pre-existing or emerging alterations that ultimately contribute to the resistance to ADT. The acknowledged role of the tumor heterogeneity in this process is hard to dispute. However, it is difficult to address it in the context of limited biopsies, even if several are taken from the tumor. Comprehensive genomic analysis of the CTCs or free circulating tumor DNA might be a step toward identification of mutations present in heterogeneous subpopulations of tumor or its metastases.


Arabidopsis thaliana hypocotyls, in which AR initiate upon de-etiolation or in tomato seedlings, in which AR initiate upon flooding or high water availability. In the hypocotyl AR originate from a cell layer reminiscent to the pericycle in the primary root (PR) and the initiated AR share histological and developmental characteristics with lateral roots (LRs). In contrast to the PR however, the hypocotyl is a determinate structure with an established final number of cells. This points to differences between the induction of hypocotyl AR and LR on the PR, as the latter grows indeterminately. The induction of AR on the hypocotyl takes place in environmental conditions that differ from those that control LR formation. Hence, AR formation depends on differentially regulated gene products. Similarly to AR induction in stem cuttings, the capacity to induce hypocotyl AR is genotype-dependent and the plant growth regulator auxin is a key regulator controlling the rooting response. The hormones cytokinins, ethylene, jasmonic acid, and strigolactones in general reduce the root-inducing capacity. The involvement of this many regulators indicates that a tight control and fine-tuning of the initiation and emergence of AR exists. Recently, several genetic factors, specific to hypocotyl adventitious rooting in A. thaliana, have been uncovered. These factors reveal a dedicated signaling network that drives AR formation in the Arabidopsis hypocotyl. Here we provide an overview of the environmental and genetic factors controlling hypocotyl-born AR and we summarize how AR formation and the regulating factors of this organogenesis are distinct from LR induction.Wound-induced adventitious root (AR) formation is a requirement for plant survival upon root damage inflicted by pathogen attack, but also during the regeneration of plant stem cuttings for clonal propagation of elite plant varieties. Yet, adventitious rooting also takes place without wounding. This happens for example in etiolated  Plant roots, hidden below the soil surface, are generally overlooked when one thinks about plant growth and development . Roots hThe plasticity of the plant’s morphology results in a variable root entity, consisting of different root-types organized in a varying spatial configuration. In general, the root system is comprised of at least two root-types: the PR, which is formed during embryogenesis, and post-embryonically initiated lateral roots (LRs) branching of the PR . In addiArabidopsis thaliana has been undertaken , SCR (SCARECROW), and SHR (SHORT ROOT) are also involved in the maintenance of the LR stem cell niche, and most likely also in the AR meristem  emerge from undifferentiated callus or from reprogrammed cells. Hence, the cellular origin of AR is uncertain and different tissues, often associated with the vasculature or cambial layers, are proposed to generate AR founder cells . To gainsegments . In the  with LR . In conts assays , AR forms assays . Even ths assays . In thisAdventitious root formation is a complex process, influenced by multiple factors, including phytohormones, light, wounding, and stress. Auxin plays a central role  and cytoAuxin is important for many aspects of root development, including initiation and emergence, patterning of apical meristem, gravitropism, and root elongation. Auxin plays a key role in both AR and LR development . Mutantssolitary root SLR/IAA14 signal transduction factor lead to omission of cell divisions in the pericycle and aborted LR formation  family positively regulates LR formation in an auxin dependent way  and argonaute 1 (ago1 mutant showed that auxin-conjugate levels are reduced and that AUXIN RESPONSE FACTOR 17 (ARF17) is overexpressed. This suggests ARF17 has a negative role during AR formation  and activating (ARF6 and ARF8) factors is post-transcriptionally regulated by miR160 and miR167  indicates similarities between the auxin-mediated signaling pathway controlling AR development and the LR induction pathway. Moreover, gh3 mutants are not specific to AR processes only and show small enhancement of LR formation because the enzymes they encode are also involved in auxin homeostasis , shows defects of vascular development in the PR and LR but not in de novo initiated AR  mutant fails to initiate ARP in the hypocotyl, but still initiates LR in PR segments , lower numbers of LR are recorded, while in the ethylene-insensitive mutants (ein2 and Nr) more root branches are formed , the precursor of ethylene, results in increased root numbers . Also inormation , 1989. Ie formed , 2010. He formed . In A. te plants . It oftee plants . Howeverpocotyls . ConsequArabidopsis and tomato may reflect a differential adaptation to flooding. Tomato is sensitive to water-logging and moisture, and the formation of AR is started shortly after submergence  determine the rooting responses. NO is related to stress responses and NO-production is enhanced under elevated COwounding . NO is awounding . In eucaLR or AR . MoreoveLR or AR , 2004. NLR or AR . NO is eLR or AR . MoreoveLR or AR . NO-homeLR or AR . This coLR or AR . PolyamiLR or AR .The different role of plant growth regulators and signaling factors, requires a tight control of initiation and emergence fine-tuning during the development of different root-types. As hormones are translators of environmental conditions, the above mentioned differences most likely represent environmental conditions that differentiate between AR and LR formation . The plaArabidopsis, removal of the PR leads to rapid AR induction at the hypocotyl base (Whereas in some species, AR initiate spontaneously, in other species AR are only formed upon stresses such as wounding or flooding or induced by nutrient shortage. Both in plant cuttings and in intact plants, wounding-associated dedifferentiation is often a requirement to form roots . Upon PRtyl base , but AR tyl base .TIR1 (A general tendency is that roots branch more in nutrient-rich conditions . High niTIR1 . In geneTIR1 . Howeverde novo formation of ARP  or emer(Poaceae . Ethylen(Poaceae . Besides(Poaceae .A. thaliana the PR, LR, and AR together form a flexible root system that is capable to adapt to changing environmental conditions. Despite the complex mix of different root-types, physiological and genetic studies have started to pinpoint specific factors to determine root identity and to control the development of one root-type over another. We believe that LR and AR share pathways, but that both root-types also have a specific set of signaling components. Because both root-types have functional similarities and differences, the different pathways intertwine at certain positions. Although some signaling factors are shared, additional research is required to draw the complex map of all interacting factors (Figure 1. A difference in tissue origin affects the regulation of the organogenic response and the conditions in the hypocotyl pericycle differ from those in a similar cell layer in the PR. Moreover, also the formation of stem-based AR is regulated by different factors than hypocotyl AR (In  factors . On the cotyl AR . We onlyThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many current therapies for metastatic castration-resistant prostate cancer (mCRPC) are aimed at AR signaling; however, resistance to these therapies is inevitable. To personalize CRPC therapy in an individual with clinical progression despite maximal AR signaling blockade, it is important to characterize the status of AR activity within their cancer. Biopsies of bone metastases are invasive and frequently fail to yield sufficient tissue for further study. Evaluation of circulating tumor cells (CTCs) offers an alternative, minimally invasive mechanism to characterize and study late-stage disease. The goal of this study was to evaluate the utility of CTC interrogation with respect to the AR as a potential novel therapeutic biomarker in patients with mCRPC.Fifteen mL of whole blood was collected from patients with progressive, metastatic mCRPC, the mononuclear cell portion was isolated, and fluorescence-activated cell sorting (FACS) was used to isolate and evaluate CTCs. A novel protocol was optimized to use ImageStreamX to quantitatively analyze AR expression and subcellular localization within CTCs. Co-expression of AR and the proliferation marker Ki67 was also determined using ImageStreamX.We found inter-patient and intra-patient heterogeneity in expression and localization of AR. Increased AR expression and nuclear localization are associated with elevated co-expression of Ki-67, consistent with the continued role for AR in castration-resistant disease. Despite intra-patient heterogeneity, CTCs from patients with prior exposure to abiraterone had increased AR expression compared to CTCs from patients who were abiraterone-naïve.As our toolbox for targeting AR function expands, our ability to evaluate AR expression and function within tumor samples from patients with late-stage disease will likely be a critical component of the personalized management of advanced prostate cancer. AR expression and nuclear localization varies within patients and between patients; however it remains associated with markers of proliferation. This supports a molecularly diverse AR-centric pathobiology imparting castration-resistance.The online version of this article (doi:10.1186/s12967-014-0313-z) contains supplementary material, which is available to authorized users. Metastatic prostate cancer is the 2nd-leading cause of cancer mortality among American men  and is aThough most men with prostate cancer initially respond to these AR-targeted therapies, resistance is inevitable. As with other targeted therapies, alteration or modification of the target is a likely resistance mechanism and a number of such mechanisms have already been described including upregulation of AR expression, as well as mutation and deletion of the AR ligand binding domain -9. NeverCirculating tumor cells (CTCs) are emerging as a source of therapy-resistant prostate cancer material, a so-called “liquid biopsy” that could serve as a therapeutic biomarker ,13. CTCsWe sought to develop and utilize techniques that allow flexibility in translational application. The eventual goal of our research is to provide support for CTC protein interrogation as a predictive therapeutic biomarker for the personalization of mCRPC care. As AR is the pivotal molecular driver of prostate cancer evolution and progression and is the foremost therapeutic target for mCRPC, our current study focused on AR interrogation. To that end we used fluorescence activated cell sorting (FACS)-based methods allowing for protein analysis and collection of live cells. We focused our approach using ImageStreamX, which combines microscopy techniques with flow cytometry. ImageStreamX captures each cellular event in a digital photograph and calculates multiple parameters, including fluorescence intensity and location, within each captured image -27. ThisAll patients were treated at the University of Chicago for mCRPC and provided informed consent per an Institutional Review Board-approved prospective clinical protocol. All patients were progressing on their current therapy by PSA or radiologic criteria. Blood (15 mL) was collected and processed within 2 hours, including isolation of mononuclear cell fraction (buffy coat). Two cohorts were evaluated: our initial cohort of FACS-sorted CTCs evaluated by immunofluorescent microscopy, and a second cohort of 20 patients with CTCs evaluated by ImageStreamX.Patient blood was drawn into blood collection tubes (BD: 362753). Isolated mononuclear cells were stained with either an Alexa-488 conjugated EpCAM antibody  or a PE-conjugated EpCAM antibody  and a QDot800 conjugated CD45 antibody . CTCs were isolated using a MoFlo XDP flow-sorting machine to sort for EpCAM positive/CD45 negative cells.Isolated CTCs that were utilized for protein expression analysis via immunofluoresence (IF) were sorted into 8-chambered slides (LAB-TEK: 154941). CTC cells were stained with combinations of the following primary antibodies overnight and secondary antibodies: Androgen Receptor  or an Alexa-647 conjugated AR antibody , PSA (Santa Cruz 7638 1:50), PE conjugated pan-cytokeratin , DyLight 680 anti-rabbit secondary , Dylight 594 anti-goat . DAPI containing mounting media was used to coverslip slides (Vector laboratories: H1200). Slides were visualized with the Leica SP2 laser scanning confocal microscope and images were analyzed using ImageJ software.To extract and amplify DNA, sorted cells were collected into PCR tubes containing 3 μl PBS and DNA extracted using REPLI-g (Qiagen) following the manufacturer’s instructions for amplification from single cells. To sequence a portion of the AR gene, primers were used to amplify exons 7 and 8, and the forward primer used for capillary sequencing. (AR intron7 F GAGGCCACCTCCTTGTCAACCCTG and AR 3UTR R2 GGCACTGCAGAGGAGTAGTGCAGA).6 cells was added, incubated for 20 minutes, washed with PBS, and centrifuged for 10 minutes at 450 G. This was repeated twice. Anti-biotin microbeads  were added to cells at a 1:4 dilution per 107 cells for 15 min, washed with PBS, and centrifuged at 450 G. Cells were re-suspended in 500 μl PBS and CD45 depleted using AutoMACS Pro (Miltenyi) per the manufacturer’s instructions. Following depletion, cells were centrifuged for 10 minutes at 450 G, washed and fixed for 15 minutes with 3.2% Ultra Pure EM Grade Formaldehyde , and stored at 4C for up to three months (average storage time was 4 weeks). Staining was performed using EpCAM , and CD45  antibodies in the dark for 30 minutes. For intracellular staining, cells were then washed with PBS. Fix/perm buffer from FoxP3 Buffer Set  was added to cells for 30 minutes then washed off with PBS. Next, cells were washed with Perm Buffer from FoxP3 Buffer Set  and centrifuged at 450 G for 5 minutes. Cells were stained intracellularly with AR , and Ki-67  in the dark for one hour. Cells were washed with Perm Buffer and then PBS. Finally, cells were stained with FxCycle Violet  and acquired on ImageStreamX (Amnis). Single stain control with gaiting strategy for each antibody is shown in Additional file Buffy coat obtained from patients with mCRPC for ImageStreamX analysis was centrifuged at 450 G for 10 minutes and washed with PBS. Cells were blocked using FcR Blocking Reagent  for 10 minutes. One μl of Biotin anti-human CD45  per 5 × 10One million LAPC-4 cells were fixed for 15 minutes with 3.2% Ultra Pure EM Grade Formaldehyde . Fixed cells and an equal number of unfixed LAPC-4 cells were washed with 1× PBS and centrifuged for 5 minutes at 450 G. Following centrifugation, cells were stained with EpCAM  for 30 minutes at 4C then washed with PBS. Both fixed and unfixed cells were analyzed via flow cytometry. FACSDiVa Software was used for data acquisition and FlowJo software was used for analysis.LAPC-4 cells were cultured in serum starved media. On day 1, either 1 μM R1881 or 10 μM of enzalutamide was added to the cells. On day 2 cells were intracellularly stained for AR as described above.VCAP or CWR22RV1 cells were fixed for 15 minutes with 3.2% Ultra Pure EM Grade Formaldehyde . Cells were washed with PBS, centrifuged for 5 minutes, and stained for EpCAM , CD45 , AR , Ki-67  and FxCycle Violet . Cells were acquired using ImageStreamX  and analyzed using IDEAS software .A mitomycin dose response curve was performed on CWR22RV1 between 1 ug/ml to 8 ug/ml, and cells were incubated for 4 hours. The media was replaced, and the cells were incubated overnight. Cells were stained for Ki-67 and analyzed by ImageStreamX.For our initial feasibility cohort, a detection rate of 35% was postulated as the minimum threshold of feasibility given the reported detection rate for CTCs between 50 and 70%. Thus, a minimum of six of the first fifteen analyzed patients needed to have isolated and visualized CTCs to satisfy our feasibility threshold and allow the study to continue.t test was used to analyze the difference in Ki-67 expression following exposure to Mitomycin. A Wilcoxon signed-rank test was used to analyze the association between Ki-67 and AR expression, and between Ki-67 and similarity index. A Wilcoxon rank sum test was used to analyze the association between AR expression and prior exposure to abiraterone, and between similarity index and prior exposure to abiraterone. A mixed model with a random patient effect was used to analyze the difference in area between EpCAM+ and EpCAM- cells.A student’s The initial step in determining feasibility of androgen receptor (AR) characterization in CTCs involved spike-in experiments in which cells from the well-established prostate cancer cell line LAPC-4 were introduced into whole blood from healthy donors. After immunostaining for epithelial (EpCAM) and white blood cell (WBC) markers (CD45), EpCAM+/CD45- events were isolated using FACS and sorted onto slide chambers for immunofluorescence (IF) Figure A. EnucleAR gene was amplified using targeted PCR from the amplified whole genome DNA of sorted C4-2 cells and WBCs. Capillary sequencing confirmed the known A→G point mutation in C4-2 events with no detectable contamination from WBCs  staining for EpCAM, prostate specific antigen (PSA) and AR followed by visualization with fluorescence microscopy Figure B. A CTC AR. We were able to visualize AR expression, and 100% (10/10) of patients had CTCs with greater fluorescence intensity (expression) in comparison to the AR-negative DU145 cell line .To further test whether AR expression and nuclear localization may be clinically relevant, we sought to examine AR biology as it relates to prior CRPC therapy. This was a clinically diverse cohort of mCRPC patients Table , with vaTo determine if AR remains a driver of proliferation in this heterogeneous cohort of patients with mCRPC, we also examined co-expression of AR and Ki-67, a well-established marker of proliferation  supporting clinical progression through a non-AR directed mechanism. In fact, our data support that patients with prior exposure to abiraterone have higher AR protein expression and statistically unchanged AR nuclear localization compared to patients who were not exposed to abiraterone. This parallels previously reported xenograft models showing increased AR expression in prostate cancer after abiraterone ,38. Our The parameters used to identify cells of interest are of concern when identifying rare events. We chose to focus on EpCAM + cells due to the wealth of experience supporting these selection criteria. It may well be, however, that there are additional markers which could allow identification/isolation of CTCs -41, whicThe relatively small sample size of this feasibility study is a limitation. A larger prospective study in a homogeneous patient population is needed to validate quantitative AR protein characterization within CTCs as a therapeutic biomarker. Fixed cells analyzed by ImageStreamX are not suitable for further interrogation of DNA or RNA expression. This is a limitation of our protein-based analysis platform. Of note, our initial FACS-based approach does allow for DNA interrogation with specificity, although this was not the focus of this current study.There are many considerations when developing novel techniques for isolating and interrogating CTCs as potential biomarkers . One priOther groups have performed highly elegant experiments examining CTC genomics and expression for other diseases ,42. In CImageStreamX analysis could easily be adapted to evaluate for expression of AR-v7 using anti-AR-v7 antibodies . Of noteIn summary, our focus on AR protein analysis in CTCs has demonstrated that interrogation of AR expression and subcellular localization may have clinical relevance. Alterations in these metrics are associated with progression on abiraterone and increased expression of a marker of cellular proliferation. Given the continued focus on AR as a therapeutic target in mCRPC, quantitative analysis of AR expression and nuclear localization has the potential to serve as a predictive therapeutic biomarker. Further validation of our techniques, in the context of prospective clinical trials is warranted. As our ability to personalize prostate cancer management improves, streamlined CTC isolation and molecular profiling will become an integral piece in the management of advanced prostate cancer. Although we focus on AR expression for the purpose of this study involving patients with mCRPC, our quantitative multiplexing methods are adaptable for other pathways of interest within mCRPC and potentially broadly applicable to other metastatic malignancies.We provide molecular characterization of the AR signaling pathway in circulating prostate cancer cells. We have demonstrated feasibility of AR characterization, showing variation in AR expression and localization. AR expression and nuclear localization in CTCs may be a dynamic therapeutic biomarker worthy of future validation in a more homogeneous patient population in the context of an AR-targeted therapy. As methods for isolation and characterization of CTCs improve, CTCs may become established as a clinical tool to guide the management of advanced prostate cancer.


Thus, we tested the hypothesis that with acclimatization to long-term hypoxia (LTH), contractility of middle cerebral arteries (MCA) is regulated by changes in expression and activation of the specific α1-AR subtypes. We conducted experiments in MCA from adult normoxic sheep maintained near sea level (300 m) and those exposed to LTH (110 days at 3801 m). Following acclimatization to LTH, ovine MCA showed a 20% reduction  in the maximum tension achieved by 10−5 M phenylephrine (PHE). LTH-acclimatized cerebral arteries also demonstrated a statistically significant (P<0.05) inhibition of PHE-induced contractility in the presence of specific α1-AR subtype antagonists. Importantly, compared to normoxic vessels, there was significantly greater (P<0.05) α1B-AR subtype mRNA and protein levels in LTH acclimatized MCA. Also, our results demonstrate that extracellular regulated kinase 1 and 2 (ERK1/2)-mediated negative feedback regulation of PHE-induced contractility is modulated by α1B-AR subtype. Overall, in ovine MCA, LTH produces profound effects on α1-AR subtype expression and function.In response to hypoxia and other stress, the sympathetic (adrenergic) nervous system regulates arterial contractility and blood flow, partly through differential activities of the alpha1 (α One possible contribution to a decrease in cerebrovascular resistance could be a shift in the structure and/or composition of the cerebral arteries favoring larger diameters and reduced hydraulic resistance. Inconsistent with this possibility, LTH had no significant effects on average artery wall thicknesses or water content Another mechanism that could potentially contribute to the LTH associated decreased cerebrovascular resistance is that of decreased vascular tone. Responses to exogenous nitric oxide (vasodilator) released from s-nitroso-N-acetylpenicillamine, however, does not differ significantly in normoxic and LTH sheep MCA Another important influence on cerebrovascular resistance under stress is the release of vasoactive neurohormones from perivascular nerves. The largest neural component of the cerebral vasculature is adrenergic in nature 1-adrenoceptor subtypes have been reported in various cell types, little is known about their expression, physiological functions, or downstream pathways in MCA. We have shown that in adult ovine cerebral arteries all three α1-AR subtypes are present 1-AR subtypes . The present study provides a deeper understanding of cerebral arterial contractility, and provides insights into the LTH regulation of α1-AR expression.Radio-ligand binding and molecular cloning in several species have demonstrated that the α1-AR family has three structurally distinct subtypes , which are widely expressed in tissues including cerebral arteries, and have differing amino acid sequences and pharmacological properties All experimental procedures were performed within the regulations of the Animal Welfare Act, the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Care and Use Committee of Loma Linda University. Sheep were obtained from Nebeker Ranch , as previously described 2 flowed at a rate adjusted to maintain its PaO2 at ∼60 Torr, similar to the value at high altitude For every experiment, 4 to 5 sheep were used from each experimental group. A total of 30 normoxic controls and 30 LTH sheep were used for completion of the present study. Following LTH exposure, animals maintained at high altitude were transported to the Center for Perinatal Biology, Loma Linda University. Upon their arrival at the Center, we placed a tracheal catheter in the sheep through which N2HPO4; 25 NaHCO3; 1.2 MgCl2; 2.5 CaCl2; 10 glucose. The arterial rings were suspended in organ baths  that contained 10 ml of modified K-H buffer maintained at 37°C and aerated with 95% O2 and 5% CO2 (pH = 7.4). Isometric force was recorded using low compliance force transducers  with analogue to digital data collection systems and software . At the beginning of each experiment, vessels were equilibrated without tension for one hour. In view of our previous findings, following stabilization the optimum resting tension was 0.7 gram (g), as at this tension the contractility response to 122 mM KCl was maximal. The contractile response to 122 mM KCl was used to normalize for the variation in smooth muscle mass in each arterial ring max (a measure of “efficacy”), and calculated pD2 , and an index of tissue “sensitivity” or “potency”).We have described this technique in several reports 1-AR subtypes can be distinguished with pharmacological agents 1A-AR is selectively inhibited by WB  1B-AR subtype is selectively inhibited by CEC (chlor-ethyl-clonidine) 1D-AR subtype 50) for vascular adrenergic receptors. First, using a given concentration of PHE (10−9 to 10−2 M) PHE, we performed a dose-response curve. Then following washout (∼5 min) and 40 min re-equilibration, we repeated the PHE dose-response in the presence of the α1-AR subtype blockers . The arteries were homogenized with a tissue grinder in ice-cold cell lysis buffer , as described previously As noted, ovine MCA were cleaned of adventitia and the endothelium was denuded in a phosphate-free balanced salt solution (BSS) of the following composition (mM): 126 NaCl; 5 KCl; 10 HEPES; 1 MgClhttp://frodo.wi.mit.edu/primer3/). These primers . A P-value of <0.05 was considered significant.All values were calculated as means ± standard error of mean (SEM). In all cases, 50 for PHE was ∼10−5 M in both oxygenation groups. Also, the maximum tension achieved in MCA by 10−5 M PHE was reduced significantly in the LTH exposed arteries as compared to the normoxic control  the receptor sensitivity for PHE was reduced ∼20% (pD2 increased from –5.1±0.1 to –4.2±0.1). In LTH MCA, however, the α1A-AR antagonist reduced the receptor sensitivity for PHE only ∼15% (pD2 increased from –5.3±0.1 to –4.5±0.06). Importantly, in normoxic cerebral arteries 10−7 M WB reduced 10−5 M (EC50) PHE-induced contractile response ∼90% , the α1A-AR subtype antagonist (WB) reduced the PHE-induced contractility . In normnse ∼90% . While, ses ∼50% . Thus, iThe reduced role of α1-AR subtype in PHE-induced contractility may be a consequence of reduced expression. Therefore, we examined mRNA and protein levels of the α1A-AR subtype. Neither mRNA and nor protein levels of α1A-AR subtype expression were changed with LTH .−5 M CEC) reduced the receptor PHE sensitivity ∼26% (pD2 increased from –5.0±0.1 to –3.7±0.1) and 19% (pD2 increased from –5.3±0.1 to –4.3±0.1) in normoxic and LTH MCA, respectively. Additionally, 10−5 M of CEC, reduced the 10−5 M PHE induced contractility ∼85% and ∼74% in normoxic and LTH MCA, respectively .−4 PD98059), we observed a significant increase in PHE-induced contractility in both normoxic and LTH arteries (−5 PD98059) in presence of specific α1-AR antagonists. As seen in Previously, we have demonstrated that ERK1/2 act as negative regulators of PKC-induced contractile responses arteries . However−5 Y-27632), we observed essentially a complete inhibition of PHE-induced contractility in both normoxic and LTH acclimatized MCA. No significant differences were observed in the presence of the several α1-AR subtype inhibitors.As seen in The present report is an extension of our previous studies on the mechanisms by which the cerebral vasculature acclimatizes to LTH. Previously, we have demonstrated that in response to such acclimatization, cerebral blood flow is maintained to levels similar to those in a normoxic animal 1-AR subtypes, members of Gq/11 protein coupled receptors, activate PLC-β to hydrolyze phosphatidylinositol 4,5,bisphosphate to form inositol 1,4,5, tri-phosphate [InsP3] and di-acyl glyerol (DAG). InsP3 acts on its receptors in the sarcoplasmic reticulum to trigger calcium release and other calcium dependent contractility pathways. DAG with and without calcium activates the several PKC isoforms. Previously, we have demonstrated that LTH leads to a 44% reduction in InsP3 release by NE-induced α1-AR activation In the present study, by use of α1-AR specific agonist (PHE) and subtype antagonists, we examined the role of LTH in mediating the regulation of specific α1-AR subtypes. In LTH acclimatized cerebral arteries, PHE-induced contractile responses were reduced to a similar extent (∼20%), as compared to norepinephrine We also have shown that ERK1/2 negatively regulate PKC-induced contractility and play a major role in calcium-sensitization pathway 1-AR subtypes in the regulation of cerebrovascular tone and cerebral blood flow in response to LTH acclimatization is not well understood. A normal acclimatization response is associated with an increase in hematocrit, hemoglobin concentration, and capillary density along with normal cerebral blood flow. However, the regulatory pathways maintaing normal (as in sea-level control) cerebral blood flow despite reduced cardiac output are not completely understood. In recent years, studies have demonstrated that adrenergic system assumes an important role in the maintenance of the cerebral blood flow. Moreover, unlike other systemic circulatory beds, large arteries have been shown to play a crucial role in the regulation and maintenance of CBF 1-AR subtypes play a role in regulation of large cerebral arteries. Quite obviously, we yet have much to learn about α1-AR subtypes and their changing role with LTH acclimatization. A caveat of these studies concerns the relative selectivity of the purported pharmacologic agonist/antagonists for the several α1-AR subtypes, as there may be some limited interaction with other adrenergic and non adrenergic receptors. Nonetheless, in previous studies, these agents have been demonstrated to be quite selective antagonists for α1-AR subtypes The role of specific α1-AR subtypes, as well as their coupling to downstream pathways. For a mechanistic overview, 1-AR subtype-mediated mechanisms in ovine MCA. An important take home message of the present study is LTH-induced increase in α1B-AR expression and their role in ERK activation. Nonetheless, this study makes apparent the complexity of intracellular signaling pathways and raises a number of important questions. For instance, by what mechanisms does LTH increase α1B-AR expression? What is the functional significance of increase in α1B- AR with LTH? To what extent is α1B-AR-mediated ERK activation responsible for vascular growth and/or other gene expression? What other signaling pathways are involved in α1-AR-mediated contractile responses in adult ovine MCA? Also of importance, how can we explain the paradox of decreased PHE-induced contractility despite an increase or no change in α1-AR subtype levels? This suggests acclimatization to LTH may be associated with changes in the plasma membrane receptor localization or internalization, or changes in the other signal transduction pathways. Insight into these pathways and physiologic mechanisms may prove to be of great clinical value in developing therapeutic interventions to prevent and/or ameliorate the sequelae of functional dysregulation of cerebral blood flow at high altitude and other conditions associated with LTH exposure.The present study extends our understanding of the ovine adrenergic system in the contractility of MCA and ultimately regulation of the cerebral blood flow. It is a logical extension of our previous findings and illustrates a small facet of this complexity. The findings provide details regarding LTH-induced acclimatization in expression and functions of the αFigure S1Relative mRNA levels (A) Glyceraldehyde 3- Phosphate Dehydrogenase and (B) Beta Actin in normoxic and LTH cerebral arteries by real-time PCR. n = 5 sheep in each group. Values are means ± standard error of means. *Denotes P = <0.05. Fold change was relative to 18 s Ribosomal RNA. GAPDH - Glyceraldehyde 3- Phosphate Dehydrogenase; ACTB - Beta Actin.(TIFF)Click here for additional data file.Figure S2Picture of RNA integrity gel demonstrating two distinct bands of 28s and 18s RNA bands. Samples 1 to 5 represents RNA isolated from normoxic cerebral arteries, whereas samples 6 to 10 represents those from LTH acclimatized sheep.(TIFF)Click here for additional data file.


Solar dimming and wind stilling (slowdown) are two outstanding climate changes occurred in China over the last four decades. The wind stilling may have suppressed the dispersion of aerosols and amplified the impact of aerosol emission on solar dimming. However, there is a lack of long-term aerosol monitoring and associated study in China to confirm this hypothesis. Here, long-term meteorological data at weather stations combined with short-term aerosol data were used to assess this hypothesis. It was found that surface solar radiation (SSR) decreased considerably with wind stilling in heavily polluted regions at a daily scale, indicating that wind stilling can considerably amplify the aerosol extinction effect on SSR. A threshold value of 3.5 m/s for wind speed is required to effectively reduce aerosols concentration. From this SSR dependence on wind speed, we further derived proxies to quantify aerosol emission and wind stilling amplification effects on SSR variations at a decadal scale. The results show that aerosol emission accounted for approximately 20% of the typical solar dimming in China, which was amplified by approximately 20% by wind stilling. Cloudiness, aerosols and water vapor modulate the transparency of atmosphere and alter the solar radiation incident at the Earth surface , and may therefore yield profound climatic, environmental, and socioeconomic consequences1245791015We circumvented the lack of long-term AOD measurements and examined the wind-aerosol-SSR hypothesis from historic routine weather station data in China. In turn we derived aerosol effects on SSR, which were used to correlate the individual contributions of aerosol emission and wind stilling with SSR variations over the last decades in China.To clarify the role of aerosol effects on SSR variations, three sub-regions with different pollution densities have been defined according to the distribution of AOD values see . The sub−2 decade−1, respectively), and the dimming magnitude in OT was relatively small (−1.9 ± 0.1 W m−2 decade−1). The solar brightening since 1990 was only observed in SC (1.6 ± 0.5 W m−2 decade−1). However, the SSR in CE continued dimming after 1990, although the dimming rate was rather weak (−0.4 ± 0.4 W m−2 decade−1) compared with that of the “dimming” period. Throughout China, subsequent to the dimming period was a leveling-off period of SSR beginning in 1990, although earlier studies2122The decadal SSR variations in the three sub-regions were calculated with quality-controlled SSR dataU) was alleviated starting in 1990 (−0.07 ± 0.01 m s−1 decade−1 for the “brightening” period compared with −0.24 ± 0.01 m s−1 decade−1 for the “dimming” period).Wind stilling is another outstanding climate change that has occurred in China15U strength may alter the aerosol extinction effect on solar radiation. U for the three sub-regions in each specific decade. In CE, the logarithmic-like curves indicate that strong U enhances SSR in the heavily polluted region, and the inter-dependence between SSR and U is reduced in the relatively clean region (OT), which was expected. However, the dependence between SSR and U is not observed in the moderately polluted SC region, which may have been caused by the impact of clouds on SSR. Note that this effect is not excluded in the results presented in Strong wind can effectively disperse aerosols and increase SSR over locallyR) and U for all the three sub-regions  to identify clean skies. This effect is also substantiated by the relationship between U and PM2.5 concentration measured in Beijing  grades according to the standards of the United States Environmental Protection Agency (USEPA), and then averaged the U values for six AQI grades. The averaged U is approximately 3.5 m s−1, the threshold value for wind speed Uth defined above, when the air quality condition is “good”  for U > Uth implies errors in estimating decadal cloud effects on SSR because cloud properties can present decadal changes.After removing the cloud effects , a logarithmic-like relationship is observed between a non-dimensional index of SSR  with high AOD values. The difference of R between conditions of calm wind and Uth can be as high as 15% in CE , aerosol emission effect (Ra) and wind stilling amplification effect (Ru) . The three proxies are calculated based on a 5-year moving window.In term of the decadal SSR variability, the Ra+u). Similarly, the most substantial dimming trend contributed by wind stilling occurred in CE, indicating that the role of wind stilling in decadal SSR changes is dependent on the level of pollution.−2 decade−1 for CE, SC, and OT, respectively) account for about 20% of the dimming trend for the three sub-regions. The wind stilling amplification effect is a small part (about 20%) of the total direct aerosol effects, and its contribution to the dimming is only a few percent. During the “brightening” period, SSR decreased in CE (−0.4 ± 0.4 W m−2 decade−1) while increased in SC (1.6 ± 0.5 W m−2 decade−1)  see . The dirWe also analyzed the aerosol effects on the decadal variations of SSR in individual months see . Winter U required to effectively remove the aerosol effect is approximately 3.5 m s−1, which can be a useful reference for managing industrial activities and vehicles emissions in large cities, such as Beijing. In terms of decadal SSR variability, this effect together with the aerosol emission effect only accounts for approximately 20% of the observed dimming and did not dominate the SSR changes over China. It is important to note that only the direct aerosol effects are taken account in the analysis. The indirect aerosol effects, of which aerosols serve as cloud condensation nuclei, can produce additional solar dimmingWind stilling can considerably amplify solar extinction by aerosols at a daily scale. This effect in China can reduce SSR by up to 15% for heavily polluted regions and seasons. The threshold SSR variations are associated with TCC and LCC (low-level cloud cover) changes over China29SSRest is the daily estimated SSR, and the coefficients  are calibrated at each CMA station using the daily SSR, TCC, and LCC of the 1960s, when aerosol emissions were relatively lowR, %) of SSR with cloud effects excluded, using the following equation:where SSRobs is daily observed SSR, SSRest the daily estimated SSR, and where U information as follows:A long-term record of aerosol loads is typically required to quantify the contribution of aerosol effects on decadal SSR variations. Visibility is often used as a proxy of AOD in China but it mainly contains information in the horizontal direction. Here, we used the dependence of SSR on wind speed after removing the cloud effects, which is shown in Ra+u (%) is the proxy for the direct aerosol effects on SSR, Uth the threshold of U, and f  is the probability density function (pdf).where f (U) in Equation Ra) is calculated as follows:The proxies were calculated for each month in a 5-year moving window at each weather station in China. To separate the impact of aerosol emission and wind stilling, the value of fref  is the reference pdf over the period from 1961–1970.where Ru) can be calculated as follows:The proxy for wind stilling amplification effect (http://dam.itpcas.ac.cn) used in the study covers the period from 1961–2010 and includes 716 China Meteorological Administration (CMA) stations. Daily data of U (10 m height), TCC, LCC, and precipitation for the period from 1961–2009 at 629 CMA stations are from the China Meteorological Data Sharing Service System at http://old-cdc.cma.gov.cn/, which is governed by the CMA. A total of 573 stations , of which 146 days have no precipitation. It should be noted that China did not measure routine PM2.5 until 2012. The monthly AOD data were provided by a Moderate-resolution Imaging Spectroradiometer (MODIS) product  and is accessible via the Earth Observing System Data and Information System (EOSDIS) of the National Aeronautics and Space Administration (NASA).PMHow to cite this article: Lin, C. et al. Impacts of wind stilling on solar radiation variability in China. Sci. Rep.5, 15135; doi: 10.1038/srep15135 (2015).