Inhibition of signaling downstream of beta-2 adrenoceptor by propranolol in prostate cancer cells.

BACKGROUND: There is accumulating evidence that propranolol, an antagonist of beta-1 and beta-2 adrenoreceptors, extends survival of patients with prostate cancer; yet it is not known whether propranolol inhibits beta-adrenergic signaling in prostate cancer cells, or systemic effects of propranolol play the leading role in slowing down cancer progression. Recently initiated clinical studies offer a possibility to test whether administration of propranolol inhibits signaling pathways in prostate tumors, however, there is limited information on the dynamics of signaling pathways activated downstream of beta-2 adrenoreceptors in prostate cancer cells and on the inactivation of these pathways upon propranolol administration. METHODS: Western blot analysis was used to test the effects of epinephrine and propranolol on activation of protein kinase (PKA) signaling in mouse prostates and PKA, extracellular signal-regulated kinase (ERK), and protein kinase B/AKT (AKT) signaling in prostate cancer cell lines. RESULTS: In prostate cancer cell lines epinephrine induced robust phosphorylation of PKA substrates pS133CREB and pS157VASP that was evident 2 min after treatments and lasted for 3-6 h. Epinephrine induced phosphorylation of AKT in PTEN-positive 22Rv1 cells, whereas changes of constitutive AKT phosphorylation were minimal in PTEN-negative PC3, C42, and LNCaP cells. A modest short-term increase of pERK in response to epinephrine was observed in all tested cell lines. Incubation of prostate cancer cells with 10-fold molar excess of propranolol for 30 min inhibited all downstream pathways activated by epinephrine. Subjecting mice to immobilization stress induced phosphorylation of S133CREB, whereas injection of propranolol at 1.5 mg/kg prevented the stress-induced phosphorylation. CONCLUSIONS: The analysis of pS133CREB and pS157VASP allows measuring activation of PKA signaling downstream of beta-2 adrenoreceptors. Presented results on the ratio of propranolol/epinephrine and the time needed to inhibit signaling downstream of beta-2 adrenoreceptors will help to design clinical studies that examine the effects of propranolol on prostate tumors.

Signaling Pathways That Control Apoptosis in Prostate Cancer.

Prostate cancer is the second most common malignancy and the fifth leading cancer-caused death in men worldwide. Therapies that target the androgen receptor axis induce apoptosis in normal prostates and provide temporary relief for advanced disease, yet prostate cancer that acquired androgen independence (so called castration-resistant prostate cancer, CRPC) invariably progresses to lethal disease. There is accumulating evidence that androgen receptor signaling do not regulate apoptosis and proliferation in prostate epithelial cells in a cell-autonomous fashion. Instead, androgen receptor activation in stroma compartments induces expression of unknown paracrine factors that maintain homeostasis of the prostate epithelium. This paradigm calls for new studies to identify paracrine factors and signaling pathways that control the survival of normal epithelial cells and to determine which apoptosis regulatory molecules are targeted by these pathways. This review summarizes the recent progress in understanding the mechanism of apoptosis induced by androgen ablation in prostate epithelial cells with emphasis on the roles of BCL-2 family proteins and "druggable" signaling pathways that control these proteins. A summary of the clinical trials of inhibitors of anti-apoptotic signaling pathways is also provided. Evidently, better knowledge of the apoptosis regulation in prostate epithelial cells is needed to understand mechanisms of androgen-independence and implement life-extending therapies for CRPC.

Long non-coding antisense RNA HYOU1-AS is essential to human breast cancer development through competitive binding hnRNPA1 to promote HYOU1 expression.

Triple negative breast cancer (TNBC) has poor prognosis due to lack of biomarker and therapeutic target. Emerging research has revealed long noncoding RNAs (lncRNAs) are involved in breast cancer progression, but their functions and regulatory mechanisms remain poorly understood, especially in TNBC. In this study, we performed lncRNA microarray analysis of five TNBC samples and their matched normal tissues, and discovered a number of differentially expressed lncRNAs. We identified an antisense lncRNA, HYOU1-AS, which is transcribed from the opposite strand of the hypoxia up-regulated 1 (HYOU1) gene, enriched in the nucleus and highly expressed in TNBC. HYOU1-AS knockdown could inhibit the proliferation and migration of the TNBC MDA-MB-231 cells, and reduce their xenograft tumor formation in nude mice. In mechanistic studies, we found that HYOU1-AS could promote the expression of HYOU1, a proliferative gene, through competitively binding to hnRNPA1, an RNA-binding protein, to relieve its post-transcriptional inhibition of the HYOU1 mRNA. Consistently, increased HYOU1 levels correlated with poor clinical outcomes of breast cancer patients based on our study of the TCGA database. Overall, our data indicated that the lncRNA HYOU1-AS promoted TNBC progression through upregulating HYOU1.

ß2-adrenoreceptor Signaling Increases Therapy Resistance in Prostate Cancer by Upregulating MCL1.

There is accumulating evidence that continuous activation of the sympathetic nervous system due to psychosocial stress increases resistance to therapy and accelerates tumor growth via ß2-adrenoreceptor signaling (ADRB2). However, the effector mechanisms appear to be specific to tumor type. Here we show that activation of ADRB2 by epinephrine, increased in response to immobilization stress, delays the loss of MCL1 apoptosis regulator (MCL1) protein expression induced by cytotoxic drugs in prostate cancer cells; and thus, increases resistance of prostate cancer xenografts to cytotoxic therapies. The effect of epinephrine on MCL1 protein depended on protein kinase A (PKA) activity, but was independent from androgen receptor expression. Furthermore, elevated blood epinephrine levels correlated positively with an increased MCL1 protein expression in human prostate biopsies. In summary, we demonstrate that stress triggers an androgen-independent antiapoptotic signaling via the ADRB2/PKA/MCL1 pathway in prostate cancer cells. IMPLICATIONS: Presented results justify clinical studies of ADRB2 blockers as therapeutics and of MCL1 protein expression as potential biomarker predicting efficacy of apoptosis-targeting drugs in prostate cancer.

ADRB2-Targeting Therapies for Prostate Cancer.

There is accumulating evidence that ß-2 adrenergic receptor (ADRB2) signaling contributes to the progression and therapy resistance of prostate cancer, whereas availability of clinically tested ß-blocker propranolol makes this pathway especially attractive as potential therapeutic target. Yet even in tumors with active ADRB2 signaling propranolol may be ineffective. Inhibition of apoptosis is one of the major mechanisms by which activation of ADRB2 contributes to prostate cancer pathophysiology. The signaling network that controls apoptosis in prostate tumors is highly redundant, with several signaling pathways targeting a few critical apoptosis regulatory molecules. Therefore, a comprehensive analysis of ADRB2 signaling in the context of other signaling mechanisms is necessary to identify patients who will benefit from propranolol therapy. This review discusses how information on the antiapoptotic mechanisms activated by ADRB2 can guide clinical trials of ADRB2 antagonist propranolol as potential life-extending therapy for prostate cancer. To select patients for clinical trials of propranolol three classes of biomarkers are proposed. First, biomarkers of ADRB2/cAMP-dependent protein kinase (PKA) pathway activation; second, biomarkers that inform about activation of other signaling pathways unrelated to ADRB2; third, apoptosis regulatory molecules controlled by ADRB2 signaling and other survival signaling pathways.

Synthesis and PI 3-Kinase Inhibition Activity of Some Novel 2,4,6-Trisubstituted 1,3,5-Triazines.

A number of new trisubstituted triazine phosphatidylinositol 3-kinase (PI3K) inhibitors were prepared via a three-step procedure utilizing sequential nucleophilic aromatic substitution and cross-coupling reactions. All were screened as PI3K inhibitors relative to the well-characterized PI3K inhibitor, ZSTK474. The most active inhibitors prepared here were 2⁻4 times more potent than ZSTK474. A leucine linker was attached to the most active inhibitor since it would remain on any peptide-containing prodrug after cleavage by a prostate-specific antigen, and it did not prevent inhibition of protein kinase B (Akt) phosphorylation, and hence, the inhibition of PI3K by the modified inhibitor.

Synthesis and PI3 Kinase Inhibition Activity of a Wortmannin-Leucine Derivative.

Wortmannin is a potent covalent inhibitor of PI3K that shows substantial in vivo toxicity and thus is unsuitable for systemic therapeutic applications. One possible approach to minimize systemic toxicity is to generate a latent wortmannin pro-drug that will be selectively activated in target tissues. To test this approach, a wortmannin derivative with a leucine linker attached to C20 has been synthesized and tested for inhibition of PI3K activity in prostate cancer cells. Analysis of PI3K pathway inhibition by Wormannin-Leu (Wn-L) and intact Wortmannin (Wn) showed that attachment of Leu at C-20 decreased potency of PI3K pathway inhibition 10-fold compared to intact wortmannin, yet exceeded the potency of a competitive PI3K inhibitor LY294002.

Synthesis and PI3 Kinase Inhibition Activity of Some Novel Trisubstituted Morpholinopyrimidines.

A number of new substituted morpholinopyrimidines were prepared utilizing sequential nucleophilic aromatic substitution and cross-coupling reactions. One of the disubstituted pyrimidines was converted into two trisubstituted compounds which were screened as PI3K inhibitors relative to the well-characterized PI3K inhibitor ZSTK474, and were found to be 1.5⁻3-times more potent. A leucine linker was attached to the most active inhibitor since it would remain on any peptide-containing prodrug after cleavage by prostate-specific antigen, and it did not prevent inhibition of AKT phosphorylation and hence the inhibition of PI3K by the modified inhibitor.

Cytoprotective effect of neuropeptides on cancer stem cells: vasoactive intestinal peptide-induced antiapoptotic signaling.

Cancer stem cells (CSCs) are increasingly considered to be responsible for tumor initiation, metastasis and drug resistance. The drug resistance mechanisms activated in CSCs have not been thoroughly investigated. Although neuropeptides such as vasoactive intestinal peptide (VIP) can promote tumor growth and activate antiapoptotic signaling in differentiated cancer cells, it is not known whether they can activate antiapoptotic mechanisms in CSCs. The objectives of this study are to unravel the cytoprotective effects of neuropeptides and identify antiapoptotic mechanisms activated by neuropeptides in response to anticancer drug treatment in CSCs. We enriched and purified CSCs (CD44+/high/CD24-/low or CD133+ population) from breast and prostate cancer cell lines, and demonstrated their stemness phenotype. Of the several neuropeptides tested, only VIP could protect CSCs from drug-induced apoptosis. A functional correlation was found between drug-induced apoptosis and dephosphorylation of proapoptotic Bcl2 family protein BAD. Similarly, VIP-induced cytoprotection correlated with BAD phosphorylation at Ser112 in CSCs. Using pharmacological inhibitors and dominant-negative proteins, we showed that VIP-induced cytoprotection and BAD phosphorylation are mediated via both Ras/MAPK and PKA pathways in CSCs of prostate cancer LNCaP and C4-2 cells, but only PKA signaling was involved in CSCs of DUVIPR (DU145 prostate cancer cells ectopically expressing VIP receptor) and breast cancer MCF7 cells. As each of these pathways partially control BAD phosphorylation at Ser112, both have to be inhibited to block the cytoprotective effects of VIP. Furthermore, VIP is unable to protect CSCs that express phosphorylation-deficient mutant-BAD, suggesting that BAD phosphorylation is essential. Thus, antiapoptotic signaling by VIP could be one of the drug resistance mechanisms by which CSCs escape from anticancer therapies. Our findings suggest the potential usefulness of VIP receptor inhibition to eliminate CSCs, and that targeting BAD might be an attractive strategy for development of novel therapeutics.

Yin Yang 1 promotes mTORC2-mediated AKT phosphorylation.

Yin Yang 1 (YY1) regulates both gene expression and protein modifications, and has shown a proliferative role in cancers. In this study, we demonstrate that YY1 promotes AKT phosphorylation at S473, a marker of AKT activation. YY1 expression positively correlated with AKT(S473) phosphorylation in a tissue microarray and cultured cells of breast cancer, but negatively associated with the distant metastasis-free survival of 166 breast cancer patients. YY1 promotes AKT phosphorylation at S473 through direct interaction with AKT, and the AKT-binding site is mapped to the residues G201-S226 on YY1. These residues are also involved in YY1 interaction with Mdm2, Ezh2, and E1A, and thus are designated as the oncogene protein binding (OPB) domain. YY1-promoted AKT phosphorylation relies on the OPB domain but is independent of either transcriptional activity of YY1 or the activity of phosphoinositide-3-kinases. We also determine that YY1-promoted mTORC2 access to AKT leads to its phosphorylation at S473. Importantly, a peptide based on the OPB domain blocks YY1 interaction with AKT and reduces AKT phosphorylation and cell proliferation. Thus, we demonstrate for the first time that YY1 promotes mTORC2-mediated AKT activation and disrupting YY1-AKT interaction by OPB domain-based peptide may represent a potential strategy for cancer therapy.

Personalized prostate cancer therapy based on systems analysis of the apoptosis regulatory network.

Targeting the androgen receptor axis provides only temporary relief for advanced prostate cancer, which often evolves into androgen-independent disease. The wide variety of signaling mechanisms connected with the pathophysiology of androgen-independent prostate cancer poses both conceptual and practical challenges for the design of efficient therapies. Analysis of apoptosis regulation in prostate cancer suggests the potential value of a systems approach that integrates information on the topology of the antiapoptotic signaling network, the signal transduction pathways that inhibit apoptosis, and the expression of proteins of the Bcl2 family. This approach could be used to identify patients most likely to respond to treatments with drugs that inhibit the signaling pathways controlling apoptosis.

A pilot study of blood epinephrine levels and CREB phosphorylation in men undergoing prostate biopsies.

PURPOSE: In mouse models of prostate cancer, increased epinephrine levels accelerated tumor growth via the beta2-adrenoreceptor/PKA signaling pathway. It is unknown, however, whether men experience increased epinephrine levels sufficient to activate the beta2-adrenoreceptor/PKA pathway in the prostate gland. We measured epinephrine levels in blood samples collected immediately prior to prostate biopsies and measured phosphorylation of S133CREB (PKA site), S112BAD, T202/Y204ERK, and S473 Akt in prostate biopsy tissue samples. METHODS: Tissue samples and 3 ml of blood were obtained from men (n = 20) recruited from the patients scheduled for prostate biopsies. Epinephrine levels were measured by ELISA. Proteins were extracted from biopsied tissue, and protein phosphorylation was measured by Western blotting with phospho-specific antibodies. Pearson and Spearman's rank correlations were analyzed to assess relationships between blood epinephrine levels and phosphorylation of CREB, BAD, AKT, and ERK. RESULTS: Epinephrine levels above 1 nM were detected in 5 of 20 patients. A strong positive correlation was observed between increased epinephrine levels and CREB phosphorylation. In contrast, no correlation was observed between epinephrine levels and phosphorylation of ERK, BAD, or AKT. CONCLUSION: Our results suggest that increased blood epinephrine levels activate the beta2-adrenoreceptor/PKA signaling pathway in human prostate glands. These results will inform future studies to examine whether beta2-selective blockers can inhibit activation of the epinephrine/ADRB2/PKA pathway in prostate tumors of men with increased epinephrine levels and explore the use of beta2-selective blockers as adjuvant therapy for prostate cancer.

Systems modeling of anti-apoptotic pathways in prostate cancer: psychological stress triggers a synergism pattern switch in drug combination therapy.

Prostate cancer patients often have increased levels of psychological stress or anxiety, but the molecular mechanisms underlying the interaction between psychological stress and prostate cancer as well as therapy resistance have been rarely studied and remain poorly understood. Recent reports show that stress inhibits apoptosis in prostate cancer cells via epinephrine/beta2 adrenergic receptor/PKA/BAD pathway. In this study, we used experimental data on the signaling pathways that control BAD phosphorylation to build a dynamic network model of apoptosis regulation in prostate cancer cells. We then compared the predictive power of two different models with or without the role of Mcl-1, which justified the role of Mcl-1 stabilization in anti-apoptotic effects of emotional stress. Based on the selected model, we examined and quantitatively evaluated the induction of apoptosis by drug combination therapies. We predicted that the combination of PI3K inhibitor LY294002 and inhibition of BAD phosphorylation at S112 would produce the best synergistic effect among 8 interventions examined. Experimental validation confirmed the effectiveness of our predictive model. Moreover, we found that epinephrine signaling changes the synergism pattern and decreases efficacy of combination therapy. The molecular mechanisms responsible for therapeutic resistance and the switch in synergism were explored by analyzing a network model of signaling pathways affected by psychological stress. These results provide insights into the mechanisms of psychological stress signaling in therapy-resistant cancer, and indicate the potential benefit of reducing psychological stress in designing more effective therapies for prostate cancer patients.

Surgical stress delays prostate involution in mice.

Androgens control growth of prostate epithelial cells and androgen deprivation induces apoptosis, leading to prostate involution. We investigated the effects of surgical stress on prostate involution induced by androgen ablation and determined the underlying mechanisms. Androgen ablation in mice was induced by surgical castration and administration of the anti-androgenic drugs bicalutamide and MDV3100. Surgical stress was induced by sham castration under isoflurane anesthesia. Surgical stress delayed apoptosis and prostate involution induced by anti-androgenic drugs. These effects of stress were prevented by administering the selective beta2-adrenoreceptor antagonist ICI118,551 and were also blocked in BAD(3SA/WT) mice expressing phosphorylation-deficient mutant BAD3SA. These results indicate that apoptosis and prostate involution in response to androgen ablation therapy could be delayed by surgical stress via the beta2-adrenoreceptor/BAD signaling pathway. Thus, surgery could interfere with androgen ablation therapy, whereas administration of beta2-adrenoreceptor antagonists may enhance its efficacy.

Combination of the PI3K inhibitor ZSTK474 with a PSMA-targeted immunotoxin accelerates apoptosis and regression of prostate cancer.

The phosphoinositide 3-kinase (PI3K) pathway is activated in most advanced prostate cancers, yet so far treatments with PI3K inhibitors have been at best tumorostatic in preclinical cancer models and do not show significant antitumor efficacy in clinical trials. Results from tissue culture experiments in prostate cancer cells suggest that PI3K inhibitors should be combined with other cytotoxic agents; however, the general toxicity of such combinations prevents translating these experimental data into preclinical and clinical models. We investigated the emerging concept of tumor-targeted synthetic lethality in prostate cancer cells by using the pan-PI3K inhibitor ZSTK474 and the immunotoxin J591PE, a protein chimera between the single-chain variable fragment of the monoclonal antibody J591 against the prostate-specific membrane antigen (PSMA) and the truncated form of the Pseudomonas aeruginosa exotoxin A (PE38QQR). The combination of ZSTK474 and J591PE increased apoptosis within 6 hours and cell death (monitored at 24-48 hours) in the PSMA-expressing cells LNCaP, C4-2, and C4-2Luc but not in control cells that do not express PSMA (PC3 and BT549 cells). Mechanistic analysis suggested that induction of apoptosis requires Bcl-2-associated death promoter (BAD) dephosphorylation and decreased expression of myeloid leukemia cell differentiation protein 1 (MCL-1). A single injection of ZSTK474 and J591PE into engrafted prostate cancer C4-2Luc cells led to consistent and stable reduction of luminescence within 6 days. These results suggest that the combination of a PI3K inhibitor and a PSMA-targeted protein synthesis inhibitor toxin represents a promising novel strategy for advanced prostate cancer therapy that should be further investigated.

BAD dephosphorylation and decreased expression of MCL-1 induce rapid apoptosis in prostate cancer cells.

PTEN loss and constitutive activation of the PI3K signaling pathway have been associated with advanced androgen-independent prostate cancer. PTEN-deficient prostate cancer C42Luc cells survive in serum-free media and show relative resistance to apoptosis even in the presence of the PI3K inhibitor ZSTK474. Yet, when ZSTK474 is combined with the translation inhibitor cycloheximide, C42Luc cells undergo apoptosis within 6 hours. We identified dephosphorylation of BAD (Bcl2-associated death promoter) as a main apoptosis-regulatory molecule downstream from PI3K, and loss of MCL-1 (Myeloid cell leukemia -1) as a major target of cycloheximide. The combination of MCL-1 knockdown and expression of phosphorylation-deficient mutant BAD2SA is sufficient to trigger rapid apoptosis in prostate cancer cells. These results establish the mechanism for the synergistic induction of apoptosis by the combination of a PI3K inhibitor and of a protein synthesis inhibitor in PTEN-deficient prostate cancer cells.

Cooperation between Dmp1 loss and cyclin D1 overexpression in breast cancer.

Cyclin D1 is a component of the core cell-cycle machinery and is frequently overexpressed in breast cancer. It physically interacts with the tumor suppressor Dmp1 that attenuates the oncogenic signals from Ras and HER2 by inducing Arf/p53-dependent cell-cycle arrest. Currently, the biological significance of Dmp1-cyclin D1 interplay in breast cancer has not been determined. Here, we show that cyclin D1 bound to Dmp1 to activate both Arf and Ink4a promoters and, consequently, induced apoptosis or G2/M cell-cycle delay in normal cells to protect them from neoplastic transformation. The cyclin D1-induced Ink4a/Arf gene expression was dependent on Dmp1 because the induction was not detected in Dmp1-deficient or DMP1-depleted cells. Arf/Ink4a expression was increased in pre-malignant mammary glands from Dmp1(+/+);MMTV-cyclin D1 and Dmp1(+/+);MMTV-D1T286A mice but significantly down-regulated in those from Dmp1-deficient mice. Selective Dmp1 deletion was found in 21% of the MMTV-D1 and D1T286A mammary carcinomas, and the Dmp1 heterozygous status significantly accelerated mouse mammary tumorigenesis with reduced apoptosis and increased metastasis. Overall, our study reveals a pivotal role of combined Dmp1 loss and cyclin D1 overexpression in breast cancer.

Dimeric DNA Aptamer Complexes for High-capacity-targeted Drug Delivery Using pH-sensitive Covalent Linkages.

Treatment with doxorubicin (Dox) results in serious systemic toxicities that limit effectiveness for cancer treatment and cause long-term health issues for cancer patients. We identified a new DNA aptamer to prostate-specific membrane antigen (PSMA) using fixed sequences to promote Dox binding and developed dimeric aptamer complexes (DACs) for specific delivery of Dox to PSMA(+) cancer cells. DACs are stable under physiological conditions and are internalized specifically into PSMA(+) C4-2 cells with minimal uptake into PSMA-null PC3 cells. Cellular internalization of DAC was demonstrated by confocal microscopy and flow cytometry. Covalent modification of DAC with Dox (DAC-D) resulted in a complex with stoichiometry ~4:1. Dox was covalently bound in DAC-D using a reversible linker that promotes covalent attachment of Dox to genomic DNA following cell internalization. Dox was released from the DAC-D under physiological conditions with a half-life of 8 hours, sufficient for in vivo targeting. DAC-D was used to selectively deliver Dox to C4-2 cells with endosomal release and nuclear localization of Dox. DAC-D was selectively cytotoxic to C4-2 cells with similar cytotoxicity as the molar equivalent of free-Dox. In contrast, DAC-D displayed minimal cytotoxicity to PC3 cells, demonstrating the complex displays a high degree of selectivity for PSMA(+) cells. DAC-D displays specificity and stability features that may be useful for improved delivery of Dox selectively to malignant tissue in vivo.Molecular Therapy-Nucleic Acids (2013) 2, e107; doi:10.1038/mtna.2013.37; published online 16 July 2013.

Recent syntheses of PI3K/Akt/mTOR signaling pathway inhibitors.

This review focuses on the syntheses of PI3K/Akt/mTOR inhibitors that have been reported outside of the patent literature in the last 5years but is largely centered on synthetic work reported in 2011 and 2012. While focused on syntheses of inhibitors, some information on in vitro and in vivo testing of compounds is also included. Many of these reported compounds are reversible, competitive adenosine triphosphate (ATP) binding inhibitors, so given the structural similarities of many of these compounds to the adenine core, this review presents recent work on inhibitors based on where the synthetic chemistry was started, that is, inhibitor syntheses which started with purines/pyrimidines are followed by inhibitor syntheses which began with pyridines, pyrazines, azoles, and triazines then moves to inhibitors which bear no structural resemblance to adenine: liphagal, wortmannin and quercetin analogs. The review then finishes with a short section on recent syntheses of phosphotidyl inositol (PI) analogs since competitive PI binding inhibitors represent an alternative to the competitive ATP binding inhibitors which have received the most attention.