5-Fluorouracil and 5-fluorouracil-based prodrugs have been used clinically for decades to treat cancer. Their anticancer effects are most prominently ascribed to inhibition of thymidylate synthase (TS) by metabolite 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). However, 5-fluorouracil and FdUMP are subject to numerous unfavorable metabolic events that can drive undesired systemic toxicity. Our previous research on antiviral nucleotides suggested that substitution at the nucleoside 5'-carbon imposes conformational restrictions on the corresponding nucleoside monophosphates, rendering them poor substrates for productive intracellular conversion to viral polymerase-inhibiting triphosphate metabolites. Accordingly, we hypothesized that 5'-substituted analogs of FdUMP, which is uniquely active at the monophosphate stage, would inhibit TS while preventing undesirable metabolism. Free energy perturbation-derived relative binding energy calculations suggested that 5'(R)-CH3 and 5'(S)-CF3 FdUMP analogs would maintain TS potency. Herein, we report our computational design strategy, synthesis of 5'-substituted FdUMP analogs, and pharmacological assessment of TS inhibitory activity.

In our previous study, we observed that androgen deprivation therapy (ADT) may induce a compensatory increase in MAPK or JNK signaling. Here, we tested the effects of the MEK inhibitors PD0325901 and GSK1120212, ERK1/2 inhibitor GDC-0994, and the JNK inhibitor AS602801 alone and in combination with the AR inhibitor enzalutamide (ENZ) in androgen-sensitive LNCaP cells and androgen-resistant C4-2 and 22Rv1 cells. Enzalutamide combined with AS602801 synergistically killed LNCaP, C4-2, and 22Rv1 cells, and decreased migration and invasion of LNCaP and C4-2 cells. We studied the combination of enzalutamide with AS602801 in vivo using luciferase labeled LNCaP xenografts, and observed that combination of ENZ with AS602801 significantly suppressed tumor growth compared with either drug alone. Importantly, combination therapy resulted in dramatic loss of AR mRNA and protein. Surprisingly, mechanistic studies and Nanostring data suggest that AS602801 likely activates JNK signaling to induce apoptosis. Since AS602801 had sufficient safety and toxicity profile to advance from Phase I to Phase II in clinical trials, repurposing of this compound may represent an opportunity for rapid translation for clinical therapy of CRPC patients.

Androgen receptor (AR) signaling is a distinctive feature of prostate cancer (PCa) and represents a major therapeutic target for treating metastatic prostate cancer (mPCa). Thus, androgen deprivation therapy (ADT) is a first-line treatment for mPCa. Although initially highly effective as a treatment for mPC, ADT is characterized by the frequent emergence of resistance, a disease state termed castration-resistant prostate cancer (CRPC) and is generally incurable after progression to metastatic disease. Therefore, understanding the mechanisms underlying CRPC and subsequent progression to metastatic disease is critical. In our previous study, which was mainly focused on how transcriptional networks change in response to ADT and lead to metastasis, we analyzed matched pre-ADT and post-ADT tissue samples via RNAseq analysis of 40 formalin-fixed paraffin-embedded (FFPE) patient-matched pre-ADT biopsy (Bx) and post-ADT radical prostatectomy (RP) prostate cancer samples. We observed strong upregulation of components of the MAPK pathway including FOS, FOSB, and JUN, as well as downstream targets of MAPK signaling. These data suggest that ADT may induce a compensatory increase in MAPK signaling in response to the decrease in androgen signaling. Thus, we hypothesize that combination therapies targeting AR and the MAPK pathway may synergistically kill prostate cancer cells and prevent recurrence and progression to CRPC. In the current study, we have tested the effects of the MEK inhibitors PD0325901 and GSK1120212, ERK1/2 inhibitor GDC-0994, and the JNK inhibitor AS602801 alone and in combination with enzalutamide in androgen-sensitive LNCaP and MDA-PCa-2b cells. Cell viability assays indicated that enzalutamide combined with MEK and JNK inhibitors synergistically killed LNCaP and MDA-PCa-2b cells, and decreased migration and invasion of LNCaP cell more than any of the drugs alone. We therefore propose that combination therapy targeting AR and MEK and/or JNK signal pathways may be an effective treatment for recurrent prostate cancer. We are currently investigating the most promising combinations of enzalutamide with JNK inhibitors for anti-tumorigenic effects in vivo using a mouse xenograft model. Citation Format: Zhenghong Li, Carrie Qi Sun, Rebecca Arnold, John A. Petros, Carlos S. Moreno. Combination therapies to prevent resistance to androgen deprivation therapies in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 284.

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) IV1 Apr 2018MP87-10 NEW DESIGNER DRUGS FOR CASTRATION RESISTANT PROSTATE CANCER Carrie Sun, Subhasish Tapadar, David Gaul, Rebecca Arnold, Adegboyega Oyelere, and John Petros Carrie SunCarrie Sun More articles by this author , Subhasish TapadarSubhasish Tapadar More articles by this author , David GaulDavid Gaul More articles by this author , Rebecca ArnoldRebecca Arnold More articles by this author , Adegboyega OyelereAdegboyega Oyelere More articles by this author , and John PetrosJohn Petros More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2910AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Castration resistant prostate cancer is difficult to treat and new chemotherapeutic agents are needed. We have developed a series of rationally-designed drugs that combine an antiandrogen moiety with a histone deacetylase inhibitor (HDACi)pharmacophore with effectiveness against prostate cancer. The objective of this study was to determine those compounds that have activity in human prostate cancer cells that are resistant to enzalutamide. METHODS Four lead antiandrogen equipped HDACi compounds SBI-19, STR 66, STR 68 and STR 70 are analyzed. Enzalutamide-resistant LNCaP cells (LNCaP F876L) were obtained from John Norris at Duke University and grown in vitro. Increasing concentrations of each drug were compared to Enzalutamide and vehicle alone for cell kill. Evidence of cell selectivity and intracellular target engagement was obtained from selectively cytotoxic to AR(+) PCa cells and western blot probing for tubulin acetylation status respectively. In vivo experiments were performed using cell line xenografts in nude mice. RESULTS All four drugs were more effective than Enzalutamide alone at killing the enzalutamide-resistant prostate cancer cells. While Enzalutamide alone resulted in 55% fewer live cells compared to vehicle alone, the 4 novel compounds resulted in 80% fewer live cells under comparable conditions. Nearly 3-fold more cells survived treatment with 50 micro-molar Enzalutamide compared to the new drugs. The drugs were efficiently taken up by AR(+) LNCaP relative to the AR(-) DU145 PCa cells PCa cells, causing selective toxicity to the AR(+) cells. Effectiveness was also seen in vivo. CONCLUSIONS Using an in vitro model of castration resistant prostate cancer we have shown enhanced cell kill by a series of novel compounds that combine an antiandrogen moiety with a histone deacetylase inhibitor (HDACi)pharmacophore. These compounds are efficiently taken up and retained by androgen receptor positive prostate cancer cells. Because this model employs resistance that is due to a mutation in the androgen receptor it is highly clinically relevant. These dual-functional drugs may be promising for further pre-clinical analysis and may ultimately translate into novel therapeutics for lethal prostate cancer. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1191 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Carrie Sun More articles by this author Subhasish Tapadar More articles by this author David Gaul More articles by this author Rebecca Arnold More articles by this author Adegboyega Oyelere More articles by this author John Petros More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) IV1 Apr 2018MP87-01 RECURRENT MITOCHONDRIAL DNA MUTATION FOUND IN BONE METASTASES Carrie Sun, Colm Morrissey, John Petros, and Rebecca Arnold Carrie SunCarrie Sun More articles by this author , Colm MorrisseyColm Morrissey More articles by this author , John PetrosJohn Petros More articles by this author , and Rebecca ArnoldRebecca Arnold More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2901AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We have previously described a single DNA base alteration in the mitochondrially encoded ND3 gene (A10398G; Thr114Ala) that occurs as a bone metastasis-specific alteration in the majority of 13 prostate cancer patients with bone metastasis (Bone 78 (2015) 81-86). The objective of this study was to define the incidence of this single base alteration in an expanded series of patients and to determine whether the prostate primary also harbored this mutation or if it occurred de-novo in the bone using an ultra-sensitive assay. METHODS We developed a digital drop PCR (ddPCR) fluorescent assay using the RainDance platform designed to quantitatively interrogate the mitochondrial DNA (mtDNA) nucleotide position 10398 missense mutation. Clinical specimens from a rapid autopsy program included 39 patients with multiple tissues available including the prostate primary, soft tissue metastases, bone metastases and normal tissue controls. The number of DNA molecules with the wild type and mutant base were counted and mutation levels compared between normal tissue, primary tumor and metastatic sites. RESULTS Of 39 patients with bone metastasis that were evaluated, 20 (51%) had significantly increased levels of the 10398 missense mutation in the mitochondrially encoded ND3 gene compared to normal uninvolved tissue. The increase ranged from 2.5-59 fold in the bone. On average, 200,000 DNA molecules were counted from each tissue examined. Because of the ultra-sensitive nature of the ddPCR assay used, we also identified the same base alteration in 2 lymph node metastases and one adrenal metastasis. Of the 3 primary prostate tissues examined that gave adequate results, 2 did not have an increased alteration at the 10398 position compared to normal tissue and one did. Of the two prostate primaries without the mutation, neither bone metastasis had the mutation. In the patient whose prostate primary tumor had mutation (2.2=fold increase over normal tissue), the bone metastasis also had the mutation (36-fold increase over normal tissue). CONCLUSIONS In this expanded cohort of 39 patients with prostate cancer bone metastases, the majority (51%) had a substantially increased level of missense mutation at the 10398 nucleotide position of the mitochondrial genome in the bone metastasis. This mutational hot-spot is unprecedented in frequency in prostate cancer and implies a strong selective pressure for bone metastatic cells that had acquired this mutation in the ND3 gene of respiratory complex 1. A smaller number of soft-tissue metastases also demonstrated enrichment of this mutation. While the small number of primary prostate cancers available for analysis preclude any firm conclusions, the two cases without the mutation also did not have the mutation in the bone metastasis while the one with the mutation in the prostate primary demonstrated a 36-fold increase of the mutation in the bone metastasis. It is therefore possible that the mutation arises in the prostate and is selected for in the bone metastasis. This finding that the exact same missense mutation is present in over 50% of patients prostate cancer bone metastases far exceeds any somatic mutation previously reported in prostate cancer suggesting functional importance. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1187 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Carrie Sun More articles by this author Colm Morrissey More articles by this author John Petros More articles by this author Rebecca Arnold More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

was assessed by Western blot analysis and quantitative reverse transcription-PCR.To assess LASP1 function, we generated stable LASP1 knockdown and overexpression cell lines.For our cell growth assay, we used anchorage-independent growth conditions in 24 well plates coated with poly-Hema and monitored growth by WST-8 assay.To determine invasiveness, we monitored movement of cell through Matrigel coated invasion chambers.To measure tumorigenicity, in vivo tumor formation was assessed in mice xenografted with LNCaP cells overexpressing LASP1 RESULTS: Compared to low LASP1 expression, high LASP1 expression in prostate cancer is prone to early metastasis after radical prostatectomy (P¼0.0423).High LASP1 correlated with prostate cancer-specific mortality after radical prostatectomy (P<0.001).Knockdown of LASP1 in VehA (CRPC cells) and PC3 cells inhibited cell growth.Furthermore, knockdown of LASP1 in PC3 cells significantly decreased cell invasion and was accompanied by a decrease in snail and slug expression.Overexpression of LASP1 in LNCaP cells promoted cell growth.Nanog, a stemness-related marker, was increased by knockdown of LASP1 and decreased by overexpression of LASP1.No tumors in 10 mice inoculated with LNCaP mock cells grew in size, whereas all tumors in 10 mice inoculated with LASP1-overexpressing LNCaP cells grew in size.CONCLUSIONS: These results suggest that LASP1 induces tumorigenesis by promoting cell growth, invasion and stemness in prostate cancer.

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) I1 Apr 2016PD28-11 NOVEL DRUGS FOR TARGETING HISTONE DEACETYLASE INHIBITOR TO CELLS CONTAINING ANDROGEN RECEPTORS: ANALYSIS OF IN VIVO EFFECTIVENESS AGAINST HUMAN PROSTATE CANCER. Carrie Sun, Rebecca Arnold, David Gaul, Subhasish Tapadar, Berkley Gryder, Adegboyega Oyelere, and John Petros Carrie SunCarrie Sun More articles by this author , Rebecca ArnoldRebecca Arnold More articles by this author , David GaulDavid Gaul More articles by this author , Subhasish TapadarSubhasish Tapadar More articles by this author , Berkley GryderBerkley Gryder More articles by this author , Adegboyega OyelereAdegboyega Oyelere More articles by this author , and John PetrosJohn Petros More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.398AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES There are no highly effective chemotherapeutic agents for castration resistant prostate cancer, however these cancers typically have upregulated and overexpressed androgen receptor (AR). Histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs that are limited in effectiveness due to inefficient delivery to the tumors. We have therefore synthesized a series of novel bifunctional drugs that combine an androgen moiety and an HDACi. The objective of this study was to test the effectiveness of these new drugs in an in vivo model of human prostate cancer and compare to HDACi and antiandrogens alone. METHODS Athymic mice were injected subcutaneously with approximately 1 million LNCaP or LNCaP AR (LNCaP cells transfected with the androgen receptor to be high AR expressers). Mice were monitored until tumor formation was evident. The mice were divided into cohorts with equal average tumor volume and compounds and SAHA control administered using IP while enzalutamide was administered via oral gavage. Tumor volume was measured several times a week. For the androgen sensitive LNCaP cell line, all compounds were compared to the HDACi suberoylanilide hydroxamic acid (SAHA), enzalutamide and the combination of SAHA and enzalutamide. Four separate compounds differing in the linker length separating the AR-binding hydanthoin and HDAC inhibiting hydroxamate moieties, designated 17, 19, 22 and 28, were investigated. RESULTS Of the 4 compounds tested, there was minimal toxicity observed at the doses given. When tested against established LNCaP xenograft tumors at the 10 mg/kg dose, the most effective treatment was SAHA/enzalutamide. Next most effective (and similar in their effect) were enzalutamide alone, and compounds 19, 22 and 28 alone. Less effective but still different than vehicle control were SAHA alone and compound 17 alone. A subsequent study examining compounds 17, 19 and 22 at different doses demonstrated that even at a dose of 20 mg/kg compound 17 remained relatively less effective whereas compounds 19 and 22 remained relatively more effective even at the reduced dose of 5 mg/kg. When these compounds were tested at 10 mg/kg in established xenografts made from LNCaP AR cells that overexpressed the androgen receptor, they all showed similar effectiveness that was statistically significantly different from no treatment controls and indistinguishable from enzalutamide or the combination of SAHA and enzalutamide. CONCLUSIONS Our novel combination drugs are non-toxic to mice and are effective anti-cancer agents. SAHA was less effective in LNCaP model while significant differences in effectiveness were observed based on chemical structure for the antiandrogen-HDACi agents. Differences were less in cells that were engineered to overexpress the androgen receptor, a clinically relevant condition in prostate cancer. These data are the basis for continued evaluation of AR targeted HDACi therapy in prostate cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e658 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Carrie Sun More articles by this author Rebecca Arnold More articles by this author David Gaul More articles by this author Subhasish Tapadar More articles by this author Berkley Gryder More articles by this author Adegboyega Oyelere More articles by this author John Petros More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyProstate Cancer: Basic Research (I)1 Apr 2013215 MITOCHONDRIAL DNA MUTATIONS ENHANCE PROLIFERATION VIA MODULATION OF CELLULAR NITRIC OXIDE, AN EFFECT AMENABLE TO PHARMACOLOGIC MANIPULATION Qian Sun, Robert E Kalkreuter, Carrie Qi Sun, Rebecca S Arnold, and John A Petros Qian SunQian Sun Atlanta, GA More articles by this author , Robert E KalkreuterRobert E Kalkreuter Atlanta, GA More articles by this author , Carrie Qi SunCarrie Qi Sun Atlanta, GA More articles by this author , Rebecca S ArnoldRebecca S Arnold Atlanta, GA More articles by this author , and John A PetrosJohn A Petros Atlanta, GA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1595AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Mitochondrial DNA (mtDNA) mutations contribute to prostate cancer and can be either inherited or somatically acquired. The mechanisms by which mtDNA mutations exert their pro-cancer effects are under investigation. We have developed laboratory model systems to investigate these mechanisms and found nitric oxide (NO) to be produced in great excess by cancer cells that have a prostate cancer patient-derived mutation of the mitochondrial COI gene. The purpose of this study was to determine the relationship between NO levels and cancer cell proliferation and to study the effect of NO modulating drugs on both NO production and proliferation. METHODS Cytoplasmic hybrid (cybrid) cell lines were constructed that contained a robust cancer nucleus and either wild type or mutant mitochondrial DNA derived from a patient with the COI gene mutation T6124C (Met74Thr). Multiple clones were tested in fluorescence assays of NO production (DAF-FM DA, Life Technologies) and cell proliferation measured by FluoroReporter Blue (Life Technologies). Cells were treated with drugs that increase NO (aspirin, nitroprusside) or decrease NO (hemoglobin, L-NAME) or vehicle alone and NO levels and cell proliferation measured. RESULTS Cells with the mtDNA mutation proliferated faster than wild type cells. Pharmacologic treatments that decreased NO levels caused decreased proliferation. Treatment with drugs that increased NO levels caused increased proliferation at low doses and decreased proliferation at high doses, consistent with the known bi-phasic effect of NO on cell growth. Longer periods of treatment resulted in greater growth inhibitory effects. Of note, aspirin inhibited cell growth regardless of mitochondrial genotype, but the effect was more pronounced in mutant cells with a high baseline NO production. CONCLUSIONS A prostate cancer-derived MtDNA mutation causes increased cell growth via increased cellular NO production. Treatment with drugs that either increase or decrease this growth-optimal level of NO cause decreased cellular proliferation consistent with the known bi-phasic effects of NO (low concentrations being growth stimulatory and high levels growth inhibitory). The response to drug treatments varied by mtDNA mutation status. These studies suggest that therapeutic approaches targeting NO production in prostate cancer may be effective and those individuals with mtDNA mutations may be particularly sensitive to such therapies. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e88-e89 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Qian Sun Atlanta, GA More articles by this author Robert E Kalkreuter Atlanta, GA More articles by this author Carrie Qi Sun Atlanta, GA More articles by this author Rebecca S Arnold Atlanta, GA More articles by this author John A Petros Atlanta, GA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2010224 MOLECULAR CONSEQUENCES OF MITOCHONDRIAL DNA MUTATIONS IN PROSTATE CANCER Courtney Plattner, Carrie Sun, Takara Scott, Rebecca Arnold, and John Petros Courtney PlattnerCourtney Plattner More articles by this author , Carrie SunCarrie Sun More articles by this author , Takara ScottTakara Scott More articles by this author , Rebecca ArnoldRebecca Arnold More articles by this author , and John PetrosJohn Petros More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.282AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES It is now known that mutations in the mitochondrial DNA (mtDNA) are associated with prostate cancer. These mutations can be either inherited or somatically acquired and may contribute to the racial disparities in prostate cancer, with blacks exhibiting a higher incidence of inherited mtDNA mutations than whites. The objective of this study was to identify molecular consequences of mtDNA mutations in prostate cancer that may contribute to the malignant phenotype. By understanding these specific molecular alterations it is hoped that novel therapeutic agents may be developed targeted at this pathway, perhaps based on genetic testing. METHODS We introduced a single mtDNA base mutation that alters an amino acid into a human prostate cancer cell line. The ATP6 gene mutation T8993G results in the amino acid substitution Leu156Arg and hyperpolarizes the inner mitochondrial membrane. These cells (along with their wild type counterparts) were studied in vivo as nude mouse xenografts and in vivo for cell and molecular biologic alterations. Quantitative western analysis was used to measure changes in protein expression that resulted as a consequence of the mutation. Superoxide anion and hydrogen peroxide levels were measured by fluorescence using reagents dihydroethidium (DHE) and dichlorofluorescein (DCF) respectively. RESULTS The introduction of the 8993 mtDNA mutation increases tumor growth rates in nude mice and reactive oxygen production in these tumors. In the mutant cell lines there was a significant increase in the phosphorylated (active) form of focal adhesion kinase (pFAK) and fibroblast growth factor-1 (FGF1) compared to wild type prostate cancer cells. Cancer cells had significantly less PEDF (SERPINF1) protein than wild type cells. The mtDNA mutation also caused increased reactive oxygen, both superoxide and peroxide. CONCLUSIONS Mutations of the mitochondrial genome are common in clinical prostate cancer and may exert their carcinogenic effects via production of mitochondrially generated reactive oxygen. The T8993G mtDNA mutation causes prostate cancer cells to generate more ROS, and induces a coordinated change in three key cancer related proteins favoring the malignant phenotype. This suggests the potential future clinical translation of prostate cancer pharmaceuticals directed against mitochondrial ROS and/or specific proteins (FGF, pFAK, PEDF) based on mtDNA genotyping of patients or their tumors. Atlanta, GA© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e88 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Courtney Plattner More articles by this author Carrie Sun More articles by this author Takara Scott More articles by this author Rebecca Arnold More articles by this author John Petros More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 2010221 FUNCTIONAL ANALYSIS OF A RARE MUTATION OF MITOCHONDRIAL DNA FOUND IN A PROSTATE CANCER Rebecca Arnold, Adeboye Osunkoya, Sean O'Hearn, Fray Marshall, Douglas Wallace, Hong Yi, Elizabeth Wright, Carrie Sun, Les Costello, and John Petros Rebecca ArnoldRebecca Arnold Atlanta, GA More articles by this author , Adeboye OsunkoyaAdeboye Osunkoya Atlanta, GA More articles by this author , Sean O'HearnSean O'Hearn Irvine, CA More articles by this author , Fray MarshallFray Marshall Atlanta, GA More articles by this author , Douglas WallaceDouglas Wallace Irvine, CA More articles by this author , Hong YiHong Yi Atlanta, GA More articles by this author , Elizabeth WrightElizabeth Wright Atlanta, GA More articles by this author , Carrie SunCarrie Sun Atlanta, GA More articles by this author , Les CostelloLes Costello Baltimore, MD More articles by this author , and John PetrosJohn Petros Atlanta, GA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.279AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Mitochondrial DNA mutations are present in prostate cancer patients both as modulators of inherited predisposition to disease and as somatically acquired mutations in the cancer. In addition, mtDNA mutations can markedly influence metastatic capability of tumor cells in animal models. One gene in particular (cytochrome oxidase 1 [COI]) has been linked to prostate cancer (PNAS 102:719-724, 2005). We describe a rare heteroplasmic mutation of this gene in a patient with prostate cancer and the functional consequences of the mutation. METHODS We performed complete gene sequencing of the COI gene in laser capture microdissected patient prostate cancer tissue, peripheral blood and lymphoblast cell lines derived from the patient. Comparison to reference sequence and large online databases was done to ascertain the likelihood of pathogenicity. The activity of the enzyme (cytochrome oxidase) encoded by this gene was determined by dual wavelength spectrophotometry and reactive oxygen generation was determined using flow cytometric analysis of DCF-DA fluorescence. Electron microscopy of mutant and wild type prostate cancer cells was done. RESULTS The highly conserved DNA base (T) at nucleotide position 6124 of the mitochondrial genome was found to be mutated (to C) resulting in the amino acid substitution Met74Thr. This alteration resulted in a 29% decrease in activity of cytochrome oxidase (for which the COI gene constitutes the catalytic core) and a 1.75-fold increase in reactive oxygen (superoxide anion) production. The mutation was found to be heteroplasmic (existing in both mutant and wild type forms in approximately equal amounts) in the patient cancer tissue. Electron microscopy revealed substantial morphologic derangement of the mitochondria only in the presence of mutation. CONCLUSIONS A rare mutation of the mitochondrial gene previously linked to prostate cancer has been found in a patient and studied functionally and structurally in the laboratory. The mutation causes severe morphologic alteration of mitochondrial ultrastructure. The presumed link between mtDNA mutation and carcinogenesis (increased production of reactive oxygen species) has been definitively confirmed for this discrete amino acid altering mutation. The CO1 mutation induces a dysfuntional cytochrome oxidase activity resulting in an increase in ROS. These results are demonstrative of the important role of altered mitochondrial oxidative metabolism in prostate and other cancers. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetailsCited byAtala A (2018) Re: Bone Metastasis in Prostate Cancer: Recurring Mitochondrial DNA Mutation Reveals Selective Pressure Exerted by the Bone MicroenvironmentJournal of Urology, VOL. 196, NO. 3, (957-958), Online publication date: 1-Sep-2016. Volume 183Issue 4SApril 2010Page: e87 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rebecca Arnold Atlanta, GA More articles by this author Adeboye Osunkoya Atlanta, GA More articles by this author Sean O'Hearn Irvine, CA More articles by this author Fray Marshall Atlanta, GA More articles by this author Douglas Wallace Irvine, CA More articles by this author Hong Yi Atlanta, GA More articles by this author Elizabeth Wright Atlanta, GA More articles by this author Carrie Sun Atlanta, GA More articles by this author Les Costello Baltimore, MD More articles by this author John Petros Atlanta, GA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2010355 MOLECULAR MECHANISMS OF DEFB1: A NEW TUMOR SUPPRESSOR GENE IN RENAL AND PROSTATE CANCERS Carrie Sun, Rebecca Arnold, Fray Marshall, Julia Dorin, and John Petros Carrie SunCarrie Sun Atlanta, GA More articles by this author , Rebecca ArnoldRebecca Arnold Atlanta, GA More articles by this author , Fray MarshallFray Marshall Atlanta, GA More articles by this author , Julia DorinJulia Dorin Edinburgh, United Kingdom More articles by this author , and John PetrosJohn Petros Atlanta, GA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.422AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Human beta defensin-1 (DEFB1) is a new candidate tumor suppressor gene located at chromosome 8p23 and is frequently deleted in both renal and prostate cancers. There are specific promoter polymorphisms that cause decreased gene expression and there is loss of the protein product in clinical specimens in 83% of prostate and 90% of renal cancers (Lab Invest 83:501; 2003). Conversely, overexpression induces apoptosis of malignant cells (Cancer Res 66:8543; 2006.) Because of the lack of appropriate laboratory models the mechanisms of action of this tumor suppressor have been poorly studied. The objectives of this study were therefore to generate an appropriate model system in which to study the molecular consequences of DEFB1 expression and discover the cell signaling pathways relevant to the action of DEFB1 in malignant cells. METHODS Using DEFB1 null mice (Infect Immun 70:3053; 2002) we generated immortalized renal epithelial cells by transfection with large T antigen and H-Ras V12. DEFB1 was re-introduced into these cells by retroviral transfection and the DEFB1 null and wild type cells compared for growth characteristics and cell signaling pathways by RT-PCR and western analysis. RESULTS Immortalized DEFB1 null renal epithelial cells grew well in vitro and as in vivo tumor xenografts in nude mice while the reintroduction of DEFB1 resulted in poor growth, and caused cell death in culture. DEFB1 knock-out cells exhibited up regulation of HER-2/neu receptor and phospho-ERK and reintroduction of DEFB1 reversed both of these molecular alterations. CONCLUSIONS We have developed an animal model system that closely resembles the clinical finding in renal cell carcinoma of the cancer-specific loss of DEFB1 expression in malignant renal epithelial cells. The molecular consequences of the loss of the tumor suppressor DEFB1 include upregulation of the HER-2/neu receptor and increased phosphorylation of ERG to the active form. This model system will allow future mechanistic evaluation of DEFB1 as a tumor suppressor and may be used to test novel molecular targeted therapies. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e141 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Carrie Sun Atlanta, GA More articles by this author Rebecca Arnold Atlanta, GA More articles by this author Fray Marshall Atlanta, GA More articles by this author Julia Dorin Edinburgh, United Kingdom More articles by this author John Petros Atlanta, GA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of Urology1 Apr 2008ENIGMOL (2-AMINO-3,5-DIHYDROXYOCTADECANE [2S, 3S, 5S]) IS A NOVEL SPHINGOLIPID ANALOG THAT SHOWS A PROMISING EFFECT IN THE TREATMENT OF PROSTATE CANCER Harsha Ramaraju, Anatoly Bushnev, Carrie Qi Sun, Dennis C Liotta, Alfred H Merrill, and John A Petros Harsha RamarajuHarsha Ramaraju More articles by this author , Anatoly BushnevAnatoly Bushnev More articles by this author , Carrie Qi SunCarrie Qi Sun More articles by this author , Dennis C LiottaDennis C Liotta More articles by this author , Alfred H MerrillAlfred H Merrill More articles by this author , and John A PetrosJohn A Petros More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(08)60138-XAboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "ENIGMOL (2-AMINO-3,5-DIHYDROXYOCTADECANE [2S, 3S, 5S]) IS A NOVEL SPHINGOLIPID ANALOG THAT SHOWS A PROMISING EFFECT IN THE TREATMENT OF PROSTATE CANCER." The Journal of Urology, 179(4S), p. 46 © 2008 by American Urological AssociationFiguresReferencesRelatedDetails Volume 179Issue 4SApril 2008Page: 46 Advertisement Copyright & Permissions© 2008 by American Urological AssociationMetricsAuthor Information Harsha Ramaraju More articles by this author Anatoly Bushnev More articles by this author Carrie Qi Sun More articles by this author Dennis C Liotta More articles by this author Alfred H Merrill More articles by this author John A Petros More articles by this author Expand All Advertisement PDF downloadLoading ...

You have accessJournal of Urology1 Apr 2008MITOCHONDRIAL DNA MUTATION ENABLES GROWTH OF PROSTATE CANCER IN BONE BY ACTIVATING SPECIFIC SIGNALING PATHWAYS Rebecca S Arnold, Carrie Qi Sun, Jendai Richards, Ilse Coleman, Peter Nelson, Harsha Ramaraju, Leland WK Chung, Jae Lee, Fray F Marshall, and John A Petros Rebecca S ArnoldRebecca S Arnold More articles by this author , Carrie Qi SunCarrie Qi Sun More articles by this author , Jendai RichardsJendai Richards More articles by this author , Ilse ColemanIlse Coleman More articles by this author , Peter NelsonPeter Nelson More articles by this author , Harsha RamarajuHarsha Ramaraju More articles by this author , Leland WK ChungLeland WK Chung More articles by this author , Jae LeeJae Lee More articles by this author , Fray F MarshallFray F Marshall More articles by this author , and John A PetrosJohn A Petros More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(08)60303-1AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "MITOCHONDRIAL DNA MUTATION ENABLES GROWTH OF PROSTATE CANCER IN BONE BY ACTIVATING SPECIFIC SIGNALING PATHWAYS." The Journal of Urology, 179(4S), p. 104 © 2008 by American Urological AssociationFiguresReferencesRelatedDetails Volume 179Issue 4SApril 2008Page: 104 Advertisement Copyright & Permissions© 2008 by American Urological AssociationMetricsAuthor Information Rebecca S Arnold More articles by this author Carrie Qi Sun More articles by this author Jendai Richards More articles by this author Ilse Coleman More articles by this author Peter Nelson More articles by this author Harsha Ramaraju More articles by this author Leland WK Chung More articles by this author Jae Lee More articles by this author Fray F Marshall More articles by this author John A Petros More articles by this author Expand All Advertisement PDF downloadLoading ...

The overproduction of hydrogen peroxide is implicated in the development of numerous diseases1,2,3,4 and there is currently great interest in developing contrast agents that can image hydrogen peroxide in vivo. In this report, we demonstrate that nanoparticles formulated from peroxalate esters and fluorescent dyes can image hydrogen peroxide in vivo with high specificity and sensitivity. The peroxalate nanoparticles image hydrogen peroxide by undergoing a three-component chemiluminescent reaction between hydrogen peroxide, peroxalate esters and fluorescent dyes. The peroxalate nanoparticles have several attractive properties for in vivo imaging, such as tunable wavelength emission (460–630 nm), nanomolar sensitivity for hydrogen peroxide and excellent specificity for hydrogen peroxide over other reactive oxygen species. The peroxalate nanoparticles were capable of imaging hydrogen peroxide in the peritoneal cavity of mice during a lipopolysaccharide-induced inflammatory response. We anticipate numerous applications of peroxalate nanoparticles for in vivo imaging of hydrogen peroxide, given their high specificity and sensitivity and deep-tissue-imaging capability.

Reactive oxygen species (ROS) are emerging as candidate mediators of growth and angiogenesis in cancer. Increased ROS often correlates with cell growth, e.g., Ras‐transformed cells and cells treated with growth factors. While non‐transformed cells respond to growth factors/cytokines with the regulated production of ROS, tumor cells in culture frequently overproduce H2O2. We propose that NADPH oxidases (Nox) account for increased levels of ROS in some cancers. Previously, transfection of Nox1 into a prostate cancer cell line dramatically enhanced tumor growth (Arbiser et al.: PNAS 99:715–720, 2001).

To perform a series of in vivo cytotoxicity studies using a variety of doses of the comptothecin analogues 9-Aminocamptothecin (9-AC) and Irinotecan (CPT-11) with a human RCC xenograft tumor line (DU11983m). Using the subrenal capsule assay (80 nude mice) (NM-SRCA), 9-AC was evaluated at both low and high dosage levels (0.75 mg/kg and 1.25 mg/kg oral x10 doses over 12 days). Following an initial assessment of acute tumor inhibition, the study was extended to a survival assay with some cohorts receiving retreatment boluses on a once or twice weekly basis. CPT-11 was assessed at a dose of 100 mg/kg x3 over 9 days with weekly retreatment and two cohorts received 9-AC combined with Vinblastine (2.7 mg/kg) and Vinblastine alone, respectively. Tumor inhibition: tumor growth inhibition was significant (over 80%) with all cohorts receiving any camptothecin analogue and was virtually complete (>99% tumor inhibition) at the high dose 9-AC (1.25 mg/kg). Vinblastine alone achieved only moderate cytotoxic effect (46%) and induced the largest recorded cohort weight loss (toxicity). Survival analysis: the low and high dose 9-AC single agent cohorts were not significantly different; however, the CPT-11 cohort experienced maximal survival benefit. (P = 0.003) and the addition of Vinblastine did not enhance this survival advantage among the 9-AC cohorts. Control and single agent Vinblastine cohorts had the poorest survival with the treated group still surviving longer (P = 0.02). At 35 days after final assessment of acute tumor inhibition, all animals in both the control and Vinblastine alone cohorts were dead. None of the animals in any of the other cohorts (all of which had experienced a greater than 80% tumor inhibition) had died. No deaths occurred due to surgery or treatment toxicity and all deaths were deemed tumor related. CPT-11 and 9-AC produced a marked survival advantage in an orthotopic model of human advanced renal carcinoma and are identified as agents for further clinical assessment.

OBJECTIVE:To evaluate the impact of the camptothecin analogs on human TCC xenograft, both as monotherapy and in combination with cisplatin (CDDP).MATERIALS AND METHODS:Human transitional cell carcinoma (TCC) xenograft tumor line (DU4184) tested by subrenal capsule assay in 112 nude mice(NM-SRCA). CDDP and the camptothecin analogs irinotecan (CPT-11) and 9-aminocamptothecin(9-AC) were evaluated.RESULTS:Both of the camptothecin analogs showed significant short term tumor inhibition which translated into enhanced survival. Maximal tumor inhibition (>95%) was achieved when either of the camptothecin analogs was combined with CDDP with minimal host toxicity. This translated into 400% increase in median survival. While all controls were dead 39 days following tumor implantation, none of the combination treated animals had died.CONCLUSION:The combination of CDDP with these camptothecin analogs is an effective therapy against this model of advanced TCC. These observations suggest potential clinical value.