Publications by authors named "George Kulik"

27 Publications

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Signaling Pathways That Control Apoptosis in Prostate Cancer.

Cancers (Basel) 2021 Feb 24;13(5). Epub 2021 Feb 24.

Department of Life Sciences, Alfaisal University, Riyadh 11533, Saudi Arabia.

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.
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http://dx.doi.org/10.3390/cancers13050937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956765PMC
February 2021

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

Biochim Biophys Acta Mol Cell Res 2021 Apr 8;1868(4):118951. Epub 2021 Jan 8.

Key Laboratory of Saline-alkali Vegetation Ecology Restoration, Ministry of Education, College of Life Science, Northeast Forestry University, Harbin 150040, China. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbamcr.2021.118951DOI Listing
April 2021

β2-adrenoreceptor Signaling Increases Therapy Resistance in Prostate Cancer by Upregulating MCL1.

Mol Cancer Res 2020 12 14;18(12):1839-1848. Epub 2020 Sep 14.

Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina.

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 β2-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.
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http://dx.doi.org/10.1158/1541-7786.MCR-19-1037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080265PMC
December 2020

ADRB2-Targeting Therapies for Prostate Cancer.

Authors:
George Kulik

Cancers (Basel) 2019 Mar 13;11(3). Epub 2019 Mar 13.

Department of Cancer Biology, Wake Forest University Health Sciences, Medical Center Blvd, Winston-Salem, NC 27157, USA.

There is accumulating evidence that β-2 adrenergic receptor (ADRB2) signaling contributes to the progression and therapy resistance of prostate cancer, whereas availability of clinically tested β-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.
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http://dx.doi.org/10.3390/cancers11030358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468358PMC
March 2019

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

Molecules 2018 Jul 20;23(7). Epub 2018 Jul 20.

Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, USA.

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.
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http://dx.doi.org/10.3390/molecules23071791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100554PMC
July 2018

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

Molecules 2018 Jul 10;23(7). Epub 2018 Jul 10.

Department of Chemistry, Wake Forest University, P.O. Box 7486, Winston-Salem, NC 27109, USA.

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.
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http://dx.doi.org/10.3390/molecules23071675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100461PMC
July 2018

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

Molecules 2018 Jul 4;23(7). Epub 2018 Jul 4.

Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, USA.

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.
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http://dx.doi.org/10.3390/molecules23071628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100378PMC
July 2018

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

Cell Death Dis 2017 06 1;8(6):e2844. Epub 2017 Jun 1.

Laboratory of Genetic Medicine and Immunology, Weill Cornell Medicine-Qatar, Education City-Qatar Foundation, Doha 24144, Qatar.

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/CD24 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.
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http://dx.doi.org/10.1038/cddis.2017.226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520887PMC
June 2017

Yin Yang 1 promotes mTORC2-mediated AKT phosphorylation.

J Mol Cell Biol 2016 06 13;8(3):232-43. Epub 2016 Jan 13.

College of Life Science, Northeast Forestry University, Harbin, China Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA

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.
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http://dx.doi.org/10.1093/jmcb/mjw002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007621PMC
June 2016

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

Authors:
George Kulik

Asian J Androl 2015 May-Jun;17(3):471-4

Life Sciences Program, College of Science, Alfaisal University, Riyadh 11533, Saudi Arabia; Department of Cancer Biology, Wake Forest University Health Sciences, Winston Salem, NC 27157, USA, .

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.
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http://dx.doi.org/10.4103/1008-682X.143749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4430953PMC
February 2016

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

PLoS Comput Biol 2013 5;9(12):e1003358. Epub 2013 Dec 5.

Department of Radiology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America ; School of Mathematical Science, Beijing Normal University, Beijing, P. R. China.

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.
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http://dx.doi.org/10.1371/journal.pcbi.1003358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3854132PMC
August 2014

Surgical stress delays prostate involution in mice.

PLoS One 2013 6;8(11):e78175. Epub 2013 Nov 6.

Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078175PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3819334PMC
August 2014

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

Neoplasia 2013 Oct;15(10):1172-83

Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest School of Medicine, Winston-Salem, NC.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3819633PMC
http://dx.doi.org/10.1593/neo.13986DOI Listing
October 2013

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

Int Urol Nephrol 2014 Mar 21;46(3):505-10. Epub 2013 Sep 21.

Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Blvd., Winston-Salem, NC, 27157, USA.

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.
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http://dx.doi.org/10.1007/s11255-013-0513-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3959255PMC
March 2014

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

PLoS One 2013 5;8(9):e74561. Epub 2013 Sep 5.

Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074561PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3764099PMC
June 2014

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

Am J Pathol 2013 Oct 11;183(4):1339-1350. Epub 2013 Aug 11.

Department of Pathology, Wake Forest School of Medicine, Winston-Salem, North Carolina; Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina; Graduate Program in Molecular Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ajpath.2013.06.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3791682PMC
October 2013

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

Mol Ther Nucleic Acids 2013 Jul 16;2:e107. Epub 2013 Jul 16.

Department of Cancer Biology and Program in Molecular Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.

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.
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http://dx.doi.org/10.1038/mtna.2013.37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731884PMC
July 2013

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

Bioorg Med Chem 2013 Jul 9;21(14):4063-91. Epub 2013 May 9.

Department of Chemistry, Wake Forest University, PO Box 7486, Winston-Salem, NC 27109, USA.

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.
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http://dx.doi.org/10.1016/j.bmc.2013.04.083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711139PMC
July 2013

Targeting psychoemotional stress to treat prostate cancer.

Authors:
George Kulik

Asian J Androl 2013 May 15;15(3):362-3. Epub 2013 Apr 15.

Department of Cancer Biology, Department of Urology, and Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

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http://dx.doi.org/10.1038/aja.2013.30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3739661PMC
May 2013

Behavioral stress accelerates prostate cancer development in mice.

J Clin Invest 2013 Feb 25;123(2):874-86. Epub 2013 Jan 25.

Department of Cancer Biology, Wake Forest School of Medicine, Medical Center Blvd., Winston-Salem, North Carolina 27157, USA.

Prostate cancer patients have increased levels of stress and anxiety. Conversely, men who take beta blockers, which interfere with signaling from the stress hormones adrenaline and noradrenaline, have a lower incidence of prostate cancer; however, the mechanisms underlying stress-prostate cancer interactions are unknown. Here, we report that stress promotes prostate carcinogenesis in mice in an adrenaline-dependent manner. Behavioral stress inhibited apoptosis and delayed prostate tumor involution both in phosphatase and tensin homolog-deficient (PTEN-deficient) prostate cancer xenografts treated with PI3K inhibitor and in prostate tumors of mice with prostate-restricted expression of c-MYC (Hi-Myc mice) subjected to androgen ablation therapy with bicalutamide. Additionally, stress accelerated prostate cancer development in Hi-Myc mice. The effects of stress were prevented by treatment with the selective β2-adrenergic receptor (ADRB2) antagonist ICI118,551 or by inducible expression of PKA inhibitor (PKI) or of BCL2-associated death promoter (BAD) with a mutated PKA phosphorylation site (BADS112A) in xenograft tumors. Effects of stress were also blocked in Hi-Myc mice expressing phosphorylation-deficient BAD (BAD3SA). These results demonstrate interactions between prostate tumors and the psychosocial environment mediated by activation of an adrenaline/ADRB2/PKA/BAD antiapoptotic signaling pathway. Our findings could be used to identify prostate cancer patients who could benefit from stress reduction or from pharmacological inhibition of stress-induced signaling.
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http://dx.doi.org/10.1172/JCI63324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561807PMC
February 2013

Synthesis and characterization of a novel prostate cancer-targeted phosphatidylinositol-3-kinase inhibitor prodrug.

J Med Chem 2012 Sep 10;55(18):8038-46. Epub 2012 Sep 10.

Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157, USA.

The phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway is constitutively activated in a substantial proportion of prostate tumors and is considered a key mechanism supporting progression toward an androgen-independent status, for which no effective therapy is available. Therefore, PI3K inhibitors, alone or in combination with other cytotoxic drugs, could potentially be used to treat cancer with a constitutive activated PI3K/Akt pathway. To selectively target advanced prostate tumors with a constitutive activated PI3K/Akt pathway, a prostate cancer-specific PI3K inhibitor was generated by coupling the chemically modified form of the quercetin analogue LY294002 (HO-CH(2)-LY294002, compound 8) with the peptide Mu-LEHSSKLQL, in which the internal sequence HSSKLQ is a substrate for the prostate-specific antigen (PSA) protease. The result is a water-soluble and latent PI3K inhibitor prodrug (compound 11), its activation being dependent on PSA cleavage. Once activated, the L-O-CH(2)-LY294002 (compound 10) can specifically inhibit PI3K in PSA-secreting prostate cancer cells and induce apoptosis with a potency comparable to that of the original LY294002 compound.
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http://dx.doi.org/10.1021/jm300881aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738169PMC
September 2012

Expression of the Bcl-2 protein BAD promotes prostate cancer growth.

PLoS One 2009 Jul 13;4(7):e6224. Epub 2009 Jul 13.

Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA.

BAD, a pro-apoptotic protein of the Bcl-2 family, has recently been identified as an integrator of several anti-apoptotic signaling pathways in prostate cancer cells. Thus, activation of EGFR, GPCRs or PI3K pathway leads to BAD phosphorylation and inhibition of apoptosis. Increased levels of BAD in prostate carcinomas have also been reported. It appears contradictory that instead of limiting expression of pro-apoptotic protein, prostate cancer cells choose to increase BAD levels while keeping it under tight phosphorylation control. Analysis of the effect of BAD on prostate cancer xenografts has shown that increased BAD expression enhances tumor growth, while knockdown of BAD expression by shRNA inhibits tumor growth. Tissue culture experiments demonstrated that increased BAD expression stimulates proliferation of prostate cancer cells. These results suggest that increased expression of BAD provides a proliferative advantage to prostate tumors, while BAD dephosphorylation increases sensitivity of prostate cancer cells to apoptosis. Combination of proliferative and apoptotic properties prompts prostate cancer cells to be "addicted" to increased levels of phosphorylated BAD. Thus, kinases that phosphorylate BAD are plausible therapeutic targets; while monitoring BAD phosphorylation could be used to predict tumor response to treatments.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006224PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2704953PMC
July 2009

Modulation of prostate cancer genetic risk by omega-3 and omega-6 fatty acids.

J Clin Invest 2007 Jul;117(7):1866-75

Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.

Although a causal role of genetic alterations in human cancer is well established, it is still unclear whether dietary fat can modulate cancer risk in a predisposed population. Epidemiological studies suggest that diets rich in omega-3 polyunsaturated fatty acids reduce cancer incidence. To determine the influence of fatty acids on prostate cancer risk in animals with a defined genetic lesion, we used prostate-specific Pten-knockout mice, an immune-competent, orthotopic prostate cancer model, and diets with defined polyunsaturated fatty acid levels. We found that omega-3 fatty acids reduced prostate tumor growth, slowed histopathological progression, and increased survival, whereas omega-6 fatty acids had opposite effects. Introducing an omega-3 desaturase, which converts omega-6 to omega-3 fatty acids, into the Pten-knockout mice reduced tumor growth similarly to the omega-3 diet. Tumors from mice on the omega-3 diet had lower proportions of phosphorylated Bad and higher apoptotic indexes compared with those from mice on omega-6 diet. Knockdown of Bad eliminated omega-3-induced cell death, and introduction of exogenous Bad restored the sensitivity to omega-3 fatty acids. Our data suggest that modulation of prostate cancer development by polyunsaturated fatty acids is mediated in part through Bad-dependent apoptosis. This study highlights the importance of gene-diet interactions in prostate cancer.
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http://dx.doi.org/10.1172/JCI31494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1890998PMC
July 2007

Epinephrine protects cancer cells from apoptosis via activation of cAMP-dependent protein kinase and BAD phosphorylation.

J Biol Chem 2007 May 12;282(19):14094-100. Epub 2007 Mar 12.

Department of Cancer Biology, Section on Comparative Medicine, and Center for Human Genomics, Wake Forest University School of Medicine, Winston Salem, NC 27157, USA.

The stress hormone epinephrine is known to elicit multiple systemic effects that include changes in cardiovascular parameters and immune responses. However, information about its direct action on cancer cells is limited. Here we provide evidence that epinephrine reduces sensitivity of cancer cells to apoptosis through interaction with beta(2)-adrenergic receptors. The antiapoptotic mechanism of epinephrine primarily involves phosphorylation and inactivation of the proapoptotic protein BAD by cAMP-dependent protein kinase. Moreover, BAD phosphorylation was observed at epinephrine concentrations found after acute and chronic psychosocial stress. Antiapoptotic signaling by epinephrine could be one of the mechanisms by which stress promotes tumorigenesis and decreases the efficacy of anti-cancer therapies.
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http://dx.doi.org/10.1074/jbc.M611370200DOI Listing
May 2007

Epidermal growth factor protects prostate cancer cells from apoptosis by inducing BAD phosphorylation via redundant signaling pathways.

J Biol Chem 2006 Sep 17;281(37):27367-77. Epub 2006 Jul 17.

Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.

Protection from apoptosis by receptor tyrosine kinases, resistant to the inhibition of phosphatidylinositol 3 '-kinase/Akt and Ras/MEK pathways, has been reported in several cell types, including fibroblasts and epithelial prostate cancer cells; however, mechanisms of this effect were not clear. Here we report that in prostate cancer cells, epidermal growth factor activates two antiapoptotic signaling pathways that impinge on the proapoptotic protein BAD. One signaling cascade operates via the Ras/MEK module and induces BAD phosphorylation on Ser112. Another pathway predominantly relies on Rac/PAK1 signaling that leads to BAD phosphorylation on Ser136. Each of these two pathways is sufficient to protect cells from apoptosis, and therefore both have to be inhibited simultaneously to block epidermal growth factor-dependent survival. Redundancy of antiapoptotic signaling pathways should be considered when therapies targeting antiapoptotic mechanisms are designed.
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http://dx.doi.org/10.1074/jbc.M511485200DOI Listing
September 2006

Diverse antiapoptotic signaling pathways activated by vasoactive intestinal polypeptide, epidermal growth factor, and phosphatidylinositol 3-kinase in prostate cancer cells converge on BAD.

J Biol Chem 2006 Jul 25;281(30):20891-20901. Epub 2006 May 25.

Department of Cancer Biology, Wake Forest University School of Medicine, Winston Salem, North Carolina 27157. Electronic address:

It has been demonstrated that vasoactive intestinal polypeptide, epidermal growth factor, and chronic activation of phosphatidylinositol 3-kinase can protect prostate cancer cells from apoptosis; however, the signaling pathways that they use and molecules that they target are unknown. We report that vasoactive intestinal polypeptide, epidermal growth factor, and phosphatidylinositol 3-kinase activate independent signaling pathways that phosphorylate the proapoptotic protein BAD. Vasoactive intestinal polypeptide operated via protein kinase A, epidermal growth factor required Ras activity, and effects of phosphatidylinositol 3-kinase were predominantly mediated by Akt. BAD phosphorylation was critical for the antiapoptotic effects of each signaling pathway. None of these survival signals was able to rescue cells that express BAD with mutations in phosphorylation sites, whereas knockdown of BAD expression with small hairpin RNA rendered cells insensitive to apoptosis. Taken together, these results identify BAD as a convergence point of several antiapoptotic signaling pathways in prostate cells.
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http://dx.doi.org/10.1074/jbc.M602928200DOI Listing
July 2006

Smac is required for cytochrome c-induced apoptosis in prostate cancer LNCaP cells.

Cancer Res 2002 Jan;62(1):18-23

Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908, USA.

Release of cytochrome c from mitochondria to cytosol has been identified as one of the central events of apoptosis. Direct injection of cytochrome c induces apoptosis in some but not in all cell types. We observed that LNCaP prostate cancer cells failed to undergo apoptosis induced by cytochrome c microinjections. Microinjection of cytochrome c with another mitochondrial protein, Smac, was sufficient to activate caspases, however. Smac is believed to function as a neutralizer of caspase inhibitors, and mass spectrometry analysis identified XIAP as a predominant Smac binding protein in LNCaP cells. These findings are consistent with a requirement for a release of Smac from mitochondria to enable caspase activation in prostate cells. Indeed, translocation of Smac from mitochondria to cytosol was observed in LNCaP cells that undergo apoptosis and was inhibited by epidermal growth factor, which is a survival factor for these cells. These results further emphasize the central role of mitochondria in the regulation of apoptosis in prostate cancer cells.
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January 2002