Downregulation of lysine-specific demethylase 1 enhances the sensitivity of hormone-sensitive prostate cancer cells to androgen deprivation therapy

Lysine-specific demethylase 1 (LSD1) plays an important role in androgen receptor (AR) signaling, and LSD1 levels are associated with prostate cancer (PCa) progression. The present study investigated the association between the downregulation of LSD1 and the proliferation and invasiveness of PCa cells, as well as the effect of LSD1 on the androgen deprivation therapy (ADT)-induced apoptosis of PCa cells. The effect of the inhibition of LSD1 combined with ADT on PCa cell apoptosis was characterized. Furthermore, the mechanisms underlying LSD1-mediated apoptosis induced by ADT in PCa cells were investigated. Downregulation of LSD1 impaired the proliferation and invasiveness of PCa cells. Moreover, downregulation of LSD1 enhanced the apoptosis of PCa cells induced by bicalutamide in vitro. Downregulation of LSD1 decreased PSA expression, increased caspase 3 and Bax expression, decreased Bcl-2 expression and consequently enhanced castration-induced PCa cell apoptosis in vivo. These findings indicated that downregulation of LSD1 could effectively enhance the efficacy of ADT for hormone- sensitive PCa, demonstrating that this could be a promising adjunctive therapy with ADT for this disease.     

Paracrine sonic hedgehog signaling contributes significantly to acquired steroidogenesis in the prostate tumor microenvironment

Despite the substantial benefit of androgen deprivation therapy (ADT) for metastatic prostate cancer, patients often progress to castration‐resistant disease (CRPC) that is more difficult to treat. CRPC is associated with renewed androgen receptor activity in tumor cells and restoration of tumor androgen levels through acquired intratumoral steroidogenesis (AIS). Although prostate cancer (PCa) cells have been shown to have steroidogenic capability in vitro, we previously found that benign prostate stromal cells (PrSCs) can also synthesize testosterone (T) from an adrenal precursor, DHEA, when stimulated with a hedgehog (Hh) pathway agonist, SAG. Here, we show exposure of PrSCs to a different Smoothened (Smo) agonist, Ag1.5, or to conditioned medium from sonic hedgehog overexpressing LNCaP cells induces steroidogenic enzyme expression in PrSCs and significantly increases production of T and its precursor steroids in a Smo‐dependent manner from 22‐OH‐cholesterol substrate. Hh agonist‐/ligand‐treated PrSCs produced androgens at a rate similar to or greater than that of PCa cell lines. Likewise, primary bone marrow stromal cells became more steroidogenic and produced T under the influence of Smo agonist. Treatment of mice bearing LNCaP xenografts with a Smo antagonist, TAK‐441, delayed the onset of CRPC after castration and substantially reduced androgen levels in residual tumors. These outcomes support the idea that stromal cells in ADT‐treated primary or metastatic prostate tumors can contribute to AIS as a consequence of a paracrine Hh signaling microenvironment. As such, Smo antagonists may be useful for targeting prostate tumor stromal cell‐derived AIS and delaying the onset of CRPC after ADT.     

α-Viniferin activates autophagic apoptosis and cell death by reducing glucocorticoid receptor expression in castration-resistant prostate cancer cells

Prostate cancer (PCa) is one of the most commonly diagnosed urological malignancies. However, there are limited therapies for PCa patients who develop biochemical recurrence after androgen deprivation therapy (ADT). In the present study, we investigated the therapeutic efficacy and mechanism of α-Viniferin (KCV), an oligostilbene of trimeric resveratrol, against human PCa cells and found that it markedly inhibited the proliferation of LNCaP, DU145, and PC-3 cancer cells in a time- and dose-dependent manner, and had a strong cytotoxicity in non-androgen-dependent PCa cells. In addition, KCV inhibited AR downstream expression in LNCaP cells, and inhibited activation of GR signaling pathway in DU145 and PC-3. Further investigation indicated that KCV could induce cancer cell apoptosis through AMPK-mediated activation of autophagy, and inhibited GR expression in castration-resistant prostate cancer(CRPC). These findings suggest that KCV may prove to be a novel and effective therapeutic agent for the treatment of CRPC.     

Inositol hexaphosphate downregulates both constitutive and ligand‐induced mitogenic and cell survival signaling,and causes caspase‐mediated apoptotic death of human prostate carcinoma PC‐3 cells

Constitutively active mitogenic and prosurvival signaling cascades due to aberrant expression and interaction of growth factors and their receptors are well documented in human prostate cancer (PCa). Epidermal growth factor (EGF) and insulin‐like growth factor‐1 (IGF‐1) are potent mitogens that regulate proliferation and survival of PCa cells via autocrine and paracrine loops involving both mitogen‐activated protein kinase (MAPK)‐ and Akt‐mediated signaling. Accordingly, here we assessed the effect of inositol hexaphosphate (IP6) on constitutive and ligand (EGF and IGF‐1)‐induced biological responses and associated signaling cascades in advanced and androgen‐independent human PCa PC‐3 cells. Treatment of PC‐3 cells with 2 mM IP6 strongly inhibited both growth and proliferation and decreased cell viability; similar effects were also observed in other human PCa DU145 and LNCaP cells. IP6 also caused a strong apoptotic death of PC‐3 cells together with caspase 3 and PARP cleavage. Mechanistic studies showed that biological effects of IP6 were associated with inhibition of both constitutive and ligand‐induced Akt phosphorylation together with a decrease in total Akt levels, but a differential inhibitory effect on MAPKs extra cellular signal‐regulated kinase 1/2 (ERK1/2), c‐Jun N‐terminal protein kinase (JNK1/2), and p38 under constitutive and ligand‐activated conditions. Under similar condition, IP6 also inhibited AP‐1 DNA‐binding activity and decreased nuclear levels of both phospho and total c‐Fos and c‐Jun. Together, these findings for the first time establish IP6 efficacy in inhibiting aberrant EGF receptor (EGFR) or IGF‐1 receptor (IGF‐1R) pathway‐mediated sustained growth promoting and survival signaling cascades in advanced and androgen‐independent human PCa PC‐3 cells, which might have translational implications in advanced human PCa control and management. © 2009 Wiley‐Liss, Inc.     

Targeting the TLK1/NEK1 DDR axis with Thioridazine suppresses outgrowth of androgen independent prostate tumors

Standard therapy for advanced Prostate Cancer (PCa) consists of antiandrogens, which provide respite from disease progression, but ultimately fail resulting in the incurable phase of the disease: mCRPC. Targeting PCa cells before their progression to mCRPC would greatly improve the outcome. Combination therapy targeting the DNA Damage Response (DDR) has been limited by general toxicity, and a goal of clinical trials is how to target the DDR more specifically. We now show that androgen deprivation therapy (ADT) of LNCaP cells results in increased expression of TLK1B, a key kinase upstream of NEK1 and ATR and mediating the DDR that typically results in a temporary cell cycle arrest of androgen responsive PCa cells. Following DNA damage, addition of the TLK specific inhibitor, thioridazine (THD), impairs ATR and Chk1 activation, establishing the existence of a ADT > TLK1 > NEK1 > ATR > Chk1, DDR pathway, while its abrogation leads to apoptosis. Treatment with THD suppressed the outgrowth of androgen-independent (AI) colonies of LNCaP and TRAMP-C2 cells cultured with bicalutamide. Moreover, THD significantly inhibited the growth of several PCa cells in vitro (including AI lines). Administration of THD or bicalutamide was not effective at inhibiting long-term tumor growth of LNCaP xenografts. In contrast, combination therapy remarkably inhibited tumor growth via bypass of the DDR. Moreover, xenografts of LNCaP cells overexpressing a NEK1-T141A mutant were durably suppressed with bicalutamide. Collectively, these results suggest that targeting the TLK1/NEK1 axis might be a novel therapy for PCa in combination with standard of care (ADT).     

TAK‐441, a novel investigational smoothened antagonist,delays castration‐resistant progression in prostate cancer by disrupting paracrine hedgehog signaling

Hedgehog (Hh) signaling is a highly conserved intercellular and intracellular communication mechanism that governs organogenesis and is dysregulated in cancers of numerous tissues, including prostate. Up‐regulated expression of the Hh ligands, Sonic (Shh) and Desert (Dhh), has been reported in androgen‐deprived and castration‐resistant prostate cancer (CRPC). In a cohort of therapy naive, short‐ and long‐term neoadjuvant hormone therapy‐treated (NHT), and CRPC specimens, we observed elevated Dhh expression predominantly in long‐term NHT specimens and elevated Shh expression predominantly in CRPC specimens. Together with previously demonstrated reciprocal signaling between Shh‐producing prostate cancer (PCa) cells and urogenital mesenchymal fibroblasts, these results suggest that castration‐induced Hh expression promotes CRPC progression through reciprocal paracrine signaling within the tumor microenvironment. We tested whether the orally available Smoothened (Smo) antagonist, TAK‐441, could impair castration‐resistant progression of LNCaP PCa xenografts by disrupting paracrine Hh signaling. Although TAK‐441 or cyclopamine did not affect androgen withdrawal‐induced Shh up‐regulation or viability of LNCaP cells, castration‐resistant progression of LNCaP xenografts was significantly delayed in animals treated with TAK‐441. In TAK‐441‐treated xenografts, expression of murine orthologs of the Hh‐activated genes, Gli1, Gli2 and Ptch1, was substantially suppressed, while expression of the corresponding human orthologs was unaffected. As androgen‐deprived LNCaP cells up‐regulate Shh expression, but are not sensitive to Smo antagonists, these studies indicate that TAK‐441 leads to delayed castration‐resistant progression of LNCaP xenografts by disrupting paracrine Hh signaling with the tumor stroma. Thus, paracrine Hh signaling may offer unique opportunities for prognostic biomarker development, drug targeting and therapeutic response monitoring of PCa progression.     

A switch from CD44+ cell to EMT cell drives the metastasis of prostate cancer

Epithelial–mesenchymal transition (EMT) has been linked to cancer stem-like (CD44+) cell in the prostate cancer (PCa) metastasis. However, the molecular mechanism remains elusive. Here, we found EMT contributed to metastasis in PCa patients failed in androgen deprivation therapy (ADT). Castration TRAMP model also proved PCa treated with ADT promoted EMT with increased CD44+ stem-like cells. Switched CD44+ cell to EMT cell is a key step for luminal PCa cell metastasis. Our results also suggested ADT might go through promoting TGFβ1-CD44 signaling to enhance swift to EMT. Targeting CD44 with salinomycin and siRNA could inhibit cell transition and decrease PCa invasion. Together, cancer stem-like (CD44+) cells could be the initiator cells of EMT modulated by TGFβ1-CD44 signaling. Combined therapy of ADT with anti-CD44 may become a new potential therapeutic approach to battle later stage PCa.     

Increased insulin-like growth factor I receptor expression and signaling are components of androgen-independent progression in a lineage-derived prostate cancer progression model

Apoptosis and inhibition of mitosis are primary mechanisms mediating androgen ablation therapy-induced regression of prostate cancer (PCa). However, PCa readily becomes androgen independent, leading to fatal disease. Up-regulated growth and survival signaling is implicated in development of resistance to androgen ablation therapy. We are testing the hypothesis that insulin-like growth factor (IGF) responsiveness is required for androgen-independent (AI) progression. Using the LNCaP human PCa progression model, we have determined that IGF-I-mediated protection from apoptotic stress and enhanced mitotic activity is androgen dependent in LNCaP cells but is androgen independent in lineage-derived C4-2 cells. Both cell lines exhibit androgen-responsive patterns of IGF-I receptor (IGF-IR) expression, activation, and signaling to insulin receptor substrate-2 and AKT. However, C4-2 cells express higher levels of IGF-IR mRNA and protein and exhibit enhanced IGF-I-mediated phosphorylation and downstream signaling under androgen-deprived conditions. In comparisons of naive and AI metastatic human PCa specimens, we have confirmed that IGF-IR levels are elevated in advanced disease. Together with our LNCaP/C4-2 AI progression model data, these results indicate that increased IGF-IR expression is associated with AI antiapoptotic and promitotic IGF signaling in PCa disease progression.     

Interplay Between Genomic Alterations and Androgen Receptor Signaling During Prostate Cancer Development and Progression

Advanced prostate cancer (PCa) treated with androgen deprivation therapy (ADT) eventually relapses to an ADT-resistant disease referred to as castration-resistant PCa (CRPC). Recent integrative analyses of PCa genomes have led to the elucidation of potential subtypes that are revelatory to the development of PCa as well as the mechanisms of resistance to ADT and CRPC progression. These studies have confirmed that alterations in the androgen receptor (AR) signaling axis are central to CRPC progression, and have uncovered complex mechanisms by which AR and other components of the AR signaling axis affect, and are affected by, genomic changes and epigenetic transformations. Among the most frequent alterations in CRPC are direct alterations in the AR gene. These AR gene alterations include AR amplification, point mutations, and more recently AR gene rearrangements leading to expression of truncated, constitutively active AR splice variants that are impervious to ADT. In this review, we will highlight genomic alterations that are important for development and progression of PCa, with a focus on how these alterations affect, and are affected by, activity of the AR signaling axis.     

Differential regulation of metabolic pathways by androgen receptor (AR) and its constitutively active splice variant,AR-V7, in prostate cancer cells

Metastatic prostate cancer (PCa) is primarily an androgen-dependent disease, which is treated with androgen deprivation therapy (ADT). Tumors usually develop resistance (castration-resistant PCa [CRPC]), but remain androgen receptor (AR) dependent. Numerous mechanisms for AR-dependent resistance have been identified including expression of constitutively active AR splice variants lacking the hormone-binding domain. Recent clinical studies show that expression of the best-characterized AR variant, AR-V7, correlates with resistance to ADT and poor outcome. Whether AR-V7 is simply a constitutively active substitute for AR or has novel gene targets that cause unique downstream changes is unresolved. Several studies have shown that AR activation alters cell metabolism. Using LNCaP cells with inducible expression of AR-V7 as a model system, we found that AR-V7 stimulated growth, migration, and glycolysis measured by ECAR (extracellular acidification rate) similar to AR. However, further analyses using metabolomics and metabolic flux assays revealed several differences. Whereas AR increased citrate levels, AR-V7 reduced citrate mirroring metabolic shifts observed in CRPC patients. Flux analyses indicate that the low citrate is a result of enhanced utilization rather than a failure to synthesize citrate. Moreover, flux assays suggested that compared to AR, AR-V7 exhibits increased dependence on glutaminolysis and reductive carboxylation to produce some of the TCA (tricarboxylic acid cycle) metabolites. These findings suggest that these unique actions represent potential therapeutic targets.     

Survivin mediates resistance to antiandrogen therapy in prostate cancer

Resistance to antiandrogen therapy in patients with metastatic prostate cancer poses a major challenge, which, if overcome, may lead to significant advances in the treatment of these patients. Hormone resistance of prostate cancer develops, in part, from upregulation of antiapoptotic genes after androgen deprivation. Given the accumulating evidence that Survivin, a new member of the inhibitor of apoptosis (IAP) family, is associated with both cancer progression and drug resistance, we hypothesized that Survivin plays a potentially important role in hormone therapy resistance, and that targeting of Survivin may enhance sensitivity to antiandrogen therapy in prostate cancer. Patterns of Survivin expression were assessed in three prostate cancer cell lines LNCaP, PC-3, and DU-145 using quantitative Western analysis. All three cell lines were found to strongly express Survivin. In LNCaP cells with intact androgen receptors (ARs), it was observed that androgen stimulation with 5alpha-dihydrotestosterone (DHT) increased Survivin expression. Conversely, treatment with Flutamide decreased Survivin expression in LNCaP cells. We next studied the functional effect of Survivin on sensitivity to Flutamide. LNCaP cells were infected with replication-deficient adenoviruses encoding either wild-type Survivin pAd-S(WT) or a phosphorylation-defective Survivin Thr34 --> Ala dominant-negative mutant pAd-S(T34A), and then treated with Flutamide. Cell viability and apoptosis were assessed in vitro and in vivo. It was determined that Survivin can mediate resistance to such antiandrogen therapies based on our assays. Direct androgen stimulation resulted in pan-cell cycle expression of Survivin, which was found to be mediated by AKT, as it was determined that exogenous insulin-like growth factor-1 (IGF-1), a known activator of AKT signaling, could increase Survivin expression and result in pan-cell cycle expression even in AR-negative prostate cancer cell lines PC-3 and DU-145. Given this alternative mechanism of Survivin expression and our findings that Survivin can mediate resistance to Flutamide treatment, we further investigated whether IGF-1-mediated activation of Survivin via AKT could mediate resistance to antiandrogen therapy. Both in vitro and in vivo, this was found to be the case, supporting a novel mechanism of resistance to antiandrogen therapy. Our study indicates that upregulation of Survivin via IGF-1 signaling confers resistance to Flutamide in prostate cancer cells. Targeted inhibition of Survivin appears to enhance the therapeutic effects of Flutamide in vitro and in vivo, revealing a novel strategy to enhance sensitivity to androgen ablation therapy.     

MicroRNA expressions associated with progression of prostate cancer cells to antiandrogen therapy resistance

Background

Development of resistance to androgen deprivation therapy (ADT) is a major obstacle for the management of advanced prostate cancer. Therapies with androgen receptor (AR) antagonists and androgen withdrawal initially regress tumors but development of compensatory mechanisms including AR bypass signaling leads to re-growth of tumors. MicroRNAs (miRNAs) are small regulatory RNAs that are involved in maintenance of cell homeostasis but are often altered in tumor cells.

Results

In this study, we determined the association of genome wide miRNA expression (1113 unique miRNAs) with development of resistance to ADT. We used androgen sensitive prostate cancer cells that progressed to ADT and AR antagonist Casodex (CDX) resistance upon androgen withdrawal and treatment with CDX. Validation of expression of a subset of 100 miRNAs led to identification of 43 miRNAs that are significantly altered during progression of cells to treatment resistance. We also show a correlation of altered expression of 10 proteins targeted by some of these miRNAs in these cells.

Conclusions

We conclude that dynamic alterations in miRNA expression occur early on during androgen deprivation therapy, and androgen receptor blockade. The cumulative effect of these altered miRNA expression profiles is the temporal modulation of multiple signaling pathways promoting survival and acquisition of resistance. These early events are driving the transition to castration resistance and cannot be studied in already developed CRPC cell lines or tissues. Furthermore our results can be used a prognostic marker of cancers with a potential to be resistant to ADT.     

The adrenal androgen androstenediol is present in prostate cancer tissue after androgen deprivation therapy and activates mutated androgen receptor

Despite an initial response to androgen deprivation therapy, prostate cancer (PCa) progresses eventually from an androgen-dependent to an androgen-independent phenotype. One of the mechanisms of relapse is antiandrogen withdrawal phenomenon caused by mutation of 877th amino acid of androgen receptor (AR). In the present study, we established a method to measure the concentration of androstenediol (adiol) in prostate tissue. We found that adiol maintains a high concentration in PCa tissue even after androgen deprivation therapy. Furthermore, adiol is a stronger activator of mutant AR in LNCaP PCa cells and induces more cell proliferation, prostate-specific antigen (PSA) mRNA expression, and PSA promoter than dihydrotestosterone (DHT). Because antiandrogen, bicalutamide, blocked adiol activity in LNCaP cells, it was suggested that adiol effect was mediated through AR. However, high concentration of bicalutamide was necessary to block completely adiol activity. These effects were specific to LNCaP cells because adiol had less effect in PC-3 PCa cells transfected with wild-type AR than DHT and had similar effect in PC-3 cells transfected with mutant AR. The mechanism that adiol activates mutant AR in LNCaP cells did not result from the increased affinity to mutant AR or from AR's association with coactivator ARA70. However, low concentration of adiol induced more AR nuclear translocation than DHT in LNCaP cells and not PC-3 cells transfected with AR. These results indicate that adiol may cause the progression of PCa even after hormone therapy.     

IL13Rα1 prevents a castration resistant phenotype of prostate cancer by targeting hexokinase 2 for ubiquitin-mediated degradation

Objective:Androgen deprivation therapy (ADT) is still the principal treatment option for prostate cancer (PCa). In addition to reactivation of androgen receptor signaling, the resistance of PCa to apoptosis during ADT also contributes to castration resistant PCa (CRPC). A previous study reported that gene transfer of IL-13Rα2 into PCa cells sensitized the cells to the IL-13R-targeted cytotoxin IL13Rα1, leading to apoptosis. Compared with IL-13Rα2, IL13Rα1 is more constitutively expressed in PCa cells, but its function in PCa remains to be established.Methods:We determined the role and expression of IL13Rα1 in PCa cancer cells using western blotting, flow cytometry, and cell proliferation assays. Co-immunoprecipitation and mass spectrometry were used to identify the proteins that interacted with IL13Rα1, to elucidate its function.Results:In this study, we showed that IL13Rα1 was selectively suppressed in androgen-deprived PCa cells and that its suppression tended to be associated with poor prognoses of PCa patients. IL13Rα1 overexpression promoted apoptosis and inhibited tumor growth under androgen-deprived or castrated conditions (P < 0.01). Mechanistically, IL13Rα1 recruited and facilitated ubiquitin protein ligase E3C-mediated ubiquitination and degradation of hexokinase 2 (HK2), resulting in glycolytic inhibition and eventually leading to PCa cell apoptosis. Furthermore, our data revealed that mutated ataxia-telangiectasia kinase phosphorylated and facilitated the selective ubiquitin proteasome-mediated degradation of HK2. Notably, IL13Rα1-overexpressing PCa cells were more susceptible to apoptosis and exhibited reduced tumor growth after exposure to the HK2 inhibitor, 2-deoxy-D-glucose (P < 0.01).Conclusions:Our data identified a tumor suppressor role for IL13Rα1 in preventing the resistance of PCa cells to apoptosis during androgen deprivation by inhibiting glycolysis. IL13Rα1-mediated signaling involving HK2 may therefore provide a novel treatment target and strategy for CRPC.     

Secretogranin II is overexpressed in advanced prostate cancer and promotes the neuroendocrine differentiation of prostate cancer cells

AimIn prostate cancer (PCa), neuroendocrine differentiation (NED) is commonly observed in relapsing, hormone therapy-resistant tumours after androgen deprivation. However, the molecular mechanisms involved in the NED of PCa cells remain poorly understood. In this study, we investigated the expression of the neuroendocrine secretory protein secretogranin II (SgII) in PCa, and its potential involvement in the progression of this cancer as a granulogenic factor promoting NED.MethodsWe have examined SgII immunoreactivity in 25 benign prostate hyperplasia and 32 PCa biopsies. In vitro experiments were performed to investigate the involvement of SgII in the neuroendocrine differentiation and the proliferation of PCa cell lines.ResultsWe showed that immunoreactive SgII intensity correlates with tumour grade in PCa patients. Using the androgen-dependent lymph node cancer prostate cells (LNCaP) cells, we found that NED triggered by androgen deprivation is associated with the induction of SgII expression. In addition, forced expression of SgII in LNCaP cells implemented a regulated secretory pathway by triggering the formation of secretory granule-like structures competent for hormone storage and regulated release. Finally, we found that SgII promotes prostate cancer (CaP) cell proliferation.ConclusionThe present data show that SgII is highly expressed in advanced PCa and may contribute to the neuroendocrine differentiation by promoting the formation of secretory granules and the proliferation of PCa cells.     

Tyrphostin AG825 triggers p38 mitogen-activated protein kinase-dependent apoptosis in androgen-independent prostate cancer cells C4 and C4-2

Prostate cancer (PCa) progression is aided by abnormal autocrine growth factor loops. We screened for small cell-permeable inhibitors of receptor tyrosine kinases that could block their signaling and trigger cell death in PCa cell lines. We found that the human epidermal growth factor receptor (HER)-2/neu inhibitor tyrphostin AG825 is preferentially toxic to PCa cells that are phenotypically androgen independent. These effects were dose and time dependent in the human LNCaP, C4, and C4-2 cell line models of progression and correlated with the inhibition of HER-2/neu phosphoactivation and its down-regulation. In addition, we show that the inhibition of HER-2/neu signaling with AG825 triggers an imbalance between extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase activation, which leads to p38-dependent apoptosis. Inhibition of HER-1 with Compound 56 had no effect. These findings suggest that the androgen-independent C4 and C4-2 cells can be killed by selectively inhibiting their HER-2/neu signaling pathway and provide insights into the mechanism of action of AG825 in PCa cells.     

Androgens regulate Hedgehog signalling and proliferation in androgen-dependent prostate cells

Prostate cancer (PCa) is androgen sensitive in its development and progression to metastatic disease. Hedgehog (Hh) pathway activation is important in the initiation and growth of various carcinomas including PCa. We and others have observed aberrations of Hh pathway during the progression of PCa to the castration-resistant state. The involvement of androgen signalling in Hh pathway activation, however, remains largely elusive. Here we investigate the direct role of androgen signalling on Hh pathway. We examined the effect of Dihydrosterone (DHT), antiandrogen, bicalutamide, and Hh pathway inhibitor, KAAD-cyclopamine in four human prostate cell lines (two cancerous: LNCaP, VCaP, and two normal: PNT2 and PNT2-ARm which harbours a mutant version of androgen receptor (AR) that is commonly found in LNCaP). Cell proliferation as well as Hh pathway members (SHH, IHH, DHH, GLI, PTCH) mRNA expression levels were assessed. We showed that KAAD-cyclopamine decreased cell proliferation of DHT-stimulated LNCaP, VCaP and PNT2-ARm cells. SHH expression was found to be downregulated by DHT in all AR posititve cells. The negative effect of DHT on SHH expression was counteracted when cells were treated by bicalutamide. Importantly, KAAD-cyclopamine treatment seemed to inhibit AR activity. Moreover, bicalutamide as well as KAAD-cyclopamine treatments induced GLI and PTCH expression in VCaP and PNT2-ARm. Our results suggest that Hh pathway activity can be regulated by androgen signalling. Specifically, we show that the DHT-induced inhibition of Hh pathway is AR dependent. The mutual interaction between these two pathways might be important in the regulation of cell proliferation in PCa.     

Notch3 is activated by chronic hypoxia and contributes to the progression of human prostate cancer

Prostate cancer (PC) is still the second cause of cancer‐related death among men. Although patients with metastatic presentation have an ominous outcome, the vast majority of PCs are diagnosed at an early stage. Nonetheless, even among patients with clinically localized disease the outcome may vary considerably. Other than androgen sensitivity, little is known about which other signaling pathways are deranged in aggressive, localized cancers. The elucidation of such pathways may help to develop innovative therapies aimed at specific molecular targets. We report that in a hormone‐sensitive PC cell line, LNCaP, Notch3 was activated by hypoxia and sustained cell proliferation and colony formation in soft agar. Hypoxia also modulated cellular cholesterol content and the number and size of lipid rafts, causing a coalescence of small rafts into bigger clusters; under this experimental condition, Notch3 migrated from the non‐raft into the raft compartment where it colocalized with the γ‐secretase complex. We also looked at human PC biopsies and found that expression of Notch3 positively correlated with Gleason score and with expression of carbonic anhydrase IX, a marker of hypoxia. In conclusion, hypoxia triggers the activation of Notch3, which, in turn, sustains proliferation of PC cells. Notch3 pathway represents a promising target for adjuvant therapy in patients with PC.     

A novel dietary flavonoid fisetin inhibits androgen receptor signaling and tumor growth in athymic nude mice

Androgen receptor (AR)-mediated signaling plays an important role in the development and progression of prostate cancer (PCa). Hormonal therapies, mainly with combinations of antiandrogens and androgen deprivation, are the mainstay treatment for advanced disease. However, emergence of androgen resistance largely due to inefficient antihormone action limits their therapeutic usefulness. Here, we report that fisetin, a novel dietary flavonoid, acts as a novel AR ligand by competing with the high-affinity androgen to interact with the ligand binding domain of AR. We show that this physical interaction results in substantial decrease in AR stability and decrease in amino-terminal/carboxyl-terminal (N-C) interaction of AR. This results in blunting of AR-mediated transactivation of target genes including prostate-specific antigen (PSA). In addition, treatment of LNCaP cells with fisetin decreased AR protein levels, in part, by decreasing its promoter activity and by accelerating its degradation. Fisetin also synergized with Casodex in inducing apoptosis in LNCaP cells. Treatment with fisetin in athymic nude mice implanted with AR-positive CWR22Rupsilon1 human PCa cells resulted in inhibition of tumor growth and reduction in serum PSA levels. These data identify fisetin as an inhibitor of AR signaling axis and suggest that it could be a useful chemopreventive and chemotherapeutic agent to delay progression of PCa.     

Characterization of FGFR signaling in prostate cancer stem cells and inhibition via TKI treatment

Metastatic castrate-resistant prostate cancer (CRPC) remains uncurable and novel therapies are needed to better treat patients. Aberrant Fibroblast Growth Factor Receptor (FGFR) signaling has been implicated in advanced prostate cancer (PCa), and FGFR1 is suggested to be a promising therapeutic target along with current androgen deprivation therapy. We established a novel in vitro 3D culture system to study endogenous FGFR signaling in a rare subpopulation of prostate cancer stem cells (CSCs) in the cell lines PC3, DU145, LNCaP, and the induced pluripotent iPS87 cell line. 3D-propagation of PCa cells generated spheroids with increased stemness markers ALDH7A1 and OCT4, while inhibition of FGFR signaling by BGJ398 or Dovitinib decreased cell survival and proliferation of 3D spheroids. The 3D spheroids exhibited altered expression of EMT markers associated with metastasis such as E-cadherin, vimentin and Snail, compared to 2D monolayer cells. TKI treatment did not result in significant changes of EMT markers, however, specific inhibition of FGFR signaling by BGJ398 showed more favorable molecular-level changes than treatment with the multi-RTK inhibitor Dovitinib. This study provides evidence for the first time that FGFR1 plays an essential role in the proliferation of PCa CSCs at a molecular and cellular level, and suggests that TKI targeting of FGFR signaling may be a promising strategy for AR-independent CRPC.