A missense SNP in the tumor suppressor SETD2 reduces H3K36me3 and mitotic spindle integrity in Drosophila.

Mutations in SETD2 are among the most prevalent drivers of renal cell carcinoma (RCC). We identified a novel single nucleotide polymorphism (SNP) in SETD2, E902Q, within a subset of RCC patients, which manifests as both an inherited or tumor-associated somatic mutation. To determine if the SNP is biologically functional, we used CRISPR-based genome editing to generate the orthologous mutation within the Drosophila melanogaster Set2 gene. In Drosophila, the homologous amino acid substitution, E741Q, reduces H3K36me3 levels comparable to Set2 knockdown, and this loss is rescued by reintroduction of a wild-type Set2 transgene. We similarly uncovered significant defects in spindle morphogenesis, consistent with the established role of SETD2 in methylating α-Tubulin during mitosis to regulate microtubule dynamics and maintain genome stability. These data indicate the Set2 E741Q SNP affects both histone methylation and spindle integrity. Moreover, this work further suggests the SETD2 E902Q SNP may hold clinical relevance.

Discovery of 5'-Substituted 5-Fluoro-2'-deoxyuridine Monophosphate Analogs: A Novel Class of Thymidylate Synthase Inhibitors.

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.

Urine-Based Metabolomics and Machine Learning Reveals Metabolites Associated with Renal Cell Carcinoma Stage.

Urine metabolomics profiling has potential for non-invasive RCC staging, in addition to providing metabolic insights into disease progression. In this study, we utilized liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), and machine learning (ML) for the discovery of urine metabolites associated with RCC progression. Two machine learning questions were posed in the study: Binary classification into early RCC (stage I and II) and advanced RCC stages (stage III and IV), and RCC tumor size estimation through regression analysis. A total of 82 RCC patients with known tumor size and metabolomic measurements were used for the regression task, and 70 RCC patients with complete tumor-nodes-metastasis (TNM) staging information were used for the classification tasks under ten-fold cross-validation conditions. A voting ensemble regression model consisting of elastic net, ridge, and support vector regressor predicted RCC tumor size with a R2 value of 0.58. A voting classifier model consisting of random forest, support vector machines, logistic regression, and adaptive boosting yielded an AUC of 0.96 and an accuracy of 87%. Some identified metabolites associated with renal cell carcinoma progression included 4-guanidinobutanoic acid, 7-aminomethyl-7-carbaguanine, 3-hydroxyanthranilic acid, lysyl-glycine, glycine, citrate, and pyruvate. Overall, we identified a urine metabolic phenotype associated with renal cell carcinoma stage, exploring the promise of a urine-based metabolomic assay for staging this disease.

Machine Learning-Enabled Renal Cell Carcinoma Status Prediction Using Multiplatform Urine-Based Metabolomics.

Renal cell carcinoma (RCC) is diagnosed through expensive cross-sectional imaging, frequently followed by renal mass biopsy, which is not only invasive but also prone to sampling errors. Hence, there is a critical need for a noninvasive diagnostic assay. RCC exhibits altered cellular metabolism combined with the close proximity of the tumor(s) to the urine in the kidney, suggesting that urine metabolomic profiling is an excellent choice for assay development. Here, we acquired liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) data followed by the use of machine learning (ML) to discover candidate metabolomic panels for RCC. The study cohort consisted of 105 RCC patients and 179 controls separated into two subcohorts: the model cohort and the test cohort. Univariate, wrapper, and embedded methods were used to select discriminatory features using the model cohort. Three ML techniques, each with different induction biases, were used for training and hyperparameter tuning. Assessment of RCC status prediction was evaluated using the test cohort with the selected biomarkers and the optimally tuned ML algorithms. A seven-metabolite panel predicted RCC in the test cohort with 88% accuracy, 94% sensitivity, 85% specificity, and 0.98 AUC. Metabolomics Workbench Study IDs are ST001705 and ST001706.

Liver-Targeting Class I Selective Histone Deacetylase Inhibitors Potently Suppress Hepatocellular Tumor Growth as Standalone Agents.

Dysfunctions in epigenetic regulation play critical roles in tumor development and progression. Histone deacetylases (HDACs) and histone acetyl transferase (HAT) are functionally opposing epigenetic regulators, which control the expression status of tumor suppressor genes. Upregulation of HDAC activities, which results in silencing of tumor suppressor genes and uncontrolled proliferation, predominates in malignant tumors. Inhibition of the deacetylase activity of HDACs is a clinically validated cancer therapy strategy. However, current HDAC inhibitors (HDACi) have elicited limited therapeutic benefit against solid tumors. Here, we disclosed a class of HDACi that are selective for sub-class I HDACs and preferentially accumulate within the normal liver tissue and orthotopically implanted liver tumors. We observed that these compounds possess exquisite on-target effects evidenced by their induction of dose-dependent histone H4 hyperacetylation without perturbation of tubulin acetylation status and G0/G1 cell cycle arrest. Representative compounds 2 and 3a are relatively non-toxic to mice and robustly suppressed tumor growths in an orthotopic model of HCC as standalone agents. Collectively, our results suggest that these compounds may have therapeutic advantage against HCC relative to the current systemic HDACi. This prospect merits further comprehensive preclinical investigations.

Molecular characteristics and markers of advanced clear cell renal cell carcinoma: Pitfalls due to intratumoral heterogeneity and identification of genetic alterations associated with metastasis.

OBJECTIVES: To identify clear cell renal cell carcinoma-related gene mutations potentially associated with aggressive disease, sarcomatoid differentiation or poor prognosis. METHODS: We carried out genomic analysis of 217 tumor foci from 25 patients with conventional clear cell renal cell carcinoma (14 patients), clear cell renal cell carcinoma with sarcomatoid differentiation (six patients) and non-clear cell renal cell carcinoma (five patients). Each tumor nodule on the tissue block that corresponded to the same focus on the slide was separated from the normal parenchyma and other histologically distinct areas of tumor. The isolated tumor foci were used for subsequent analyses and sequencing. Deoxyribonucleic acid from the formalin-fixed paraffin-embedded tissues was extracted. Multiplex bar-coded polymerase chain reaction amplification was carried out using next-generation sequencing libraries. RESULTS: Overall, 67 protein alterations, including amino acid alterations, frame shifts and splice site mutations in seven genes were identified in the cohort of renal cell carcinoma tumors included in this study. Fewer patients with clear cell renal cell carcinoma with sarcomatoid differentiation had clear cell renal cell carcinoma-related mutations in comparison with patients with conventional clear cell renal cell carcinoma. Additionally, the average number of unique clear cell renal cell carcinoma-related protein alterations per patient was significantly lower in clear cell renal cell carcinoma with sarcomatoid differentiation than in conventional clear cell renal cell carcinoma. Mutations in PBRM1 were identified in a higher proportion of patients with high-grade tumors (World Health Organization/International Society of Urological Pathology grade 4) and in the primary tumors of six of 10 (60%) patients with metastatic disease. CONCLUSIONS: Although there are pitfalls due to intratumoral heterogeneity and sampling bias, mutations in PBRM1 may be associated with metastasis and aggressive disease in clear cell renal cell carcinoma.

The JNK inhibitor AS602801 Synergizes with Enzalutamide to Kill Prostate Cancer Cells In Vitro and In Vivo and Inhibit Androgen Receptor Expression.

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.

Discovery and mechanisms of host defense to oncogenesis: targeting the ß-defensin-1 peptide as a natural tumor inhibitor.

Human beta-defensin-1 (hBD-1) is one of a number of small cationic host-defense peptides. Besides its well-known broad-spectrum antimicrobial function, hBD-1 has recently been identified as a chromosome 8p tumor-suppressor gene. The role of hBD-1 in modulating the host immune response to oncogenesis, associated with cell signaling and potential therapeutic applications, has become increasingly appreciated over time. In this study, multiple approaches were used to illustrate hBD-1 anti-tumor activities. Results demonstrate that hBD-1 peptide alters human epidermal growth factor receptor 2 (HER2) signal transduction and represses retroviral-mediated transgene expression in cancer cells. Loss of orthologous murine defense-1 (mBD1) in mice enhances nickel sulfate-induced leiomyosarcoma and causes mouse kidney cells to exhibit increased susceptibility to HPV-16 E6/7-induced neoplastic transformation. Furthermore, for the first time, a novel function of the urine-derived hBD-1 peptide was discovered to suppress bladder cancer growth and this may lead to future applications in the treatment of malignancy.

Preoperative Metabolic Signatures of Prostate Cancer Recurrence Following Radical Prostatectomy.

Technological advances in mass spectrometry (MS), liquid chromatography (LC) separations, nuclear magnetic resonance (NMR) spectroscopy, and big data analytics have made possible studying metabolism at an "omics" or systems level. Here, we applied a multiplatform (NMR + LC-MS) metabolomics approach to the study of preoperative metabolic alterations associated with prostate cancer recurrence. Thus far, predicting which patients will recur even after radical prostatectomy has not been possible. Correlation analysis on metabolite abundances detected on serum samples collected prior to surgery from prostate cancer patients ( n = 40 remission vs n = 40 recurrence) showed significant alterations in a number of pathways, including amino acid metabolism, purine and pyrimidine synthesis, tricarboxylic acid cycle, tryptophan catabolism, glucose, and lactate. Lipidomics experiments indicated higher lipid abundances on recurrent patients for a number of classes that included triglycerides, lysophosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, diglycerides, acyl carnitines, and ceramides. Machine learning approaches led to the selection of a 20-metabolite panel from a single preoperative blood sample that enabled prediction of recurrence with 92.6% accuracy, 94.4% sensitivity, and 91.9% specificity under cross-validation conditions.

Honokiol bis-dichloroacetate (Honokiol DCA) demonstrates activity in vemurafenib-resistant melanoma in vivo.

The majority of human melanomas bears BRAF mutations and thus is treated with inhibitors of BRAF, such as vemurafenib. While patients with BRAF mutations often demonstrate an initial dramatic response to vemurafenib, relapse is extremely common. Thus, novel agents are needed for the treatment of these aggressive melanomas. Honokiol is a small molecule compound derived from Magnolia grandiflora that has activity against solid tumors and hematopoietic neoplasms. In order to increase the lipophilicity of honokiol, we have synthesized honokiol DCA, the dichloroacetate ester of honokiol. In addition, we synthesized a novel fluorinated honokiol analog, bis-trifluoromethyl-bis-(4-hydroxy-3-allylphenyl) methane (hexafluoro). Both compounds exhibited activity against A375 melanoma in vivo, but honokiol DCA was more active. Gene arrays comparing treated with vehicle control tumors demonstrated induction of the respiratory enzyme succinate dehydrogenase B (SDHB) by treatment, suggesting that our honokiol analogs induce respiration in vivo. We then examined its effect against a pair of melanomas, LM36 and LM36R, in which LM36R differs from LM36 in that LM36R has acquired vemurafenib resistance. Honokiol DCA demonstrated in vivo activity against LM36R (vemurafenib resistant) but not against parental LM36. Honokiol DCA and hexafluoro inhibited the phosphorylation of DRP1, thus stimulating a phenotype suggestive of respiration through mitochondrial normalization. Honokiol DCA may act in vemurafenib resistant melanomas to increase both respiration and reactive oxygen generation, leading to activity against aggressive melanoma in vivo.

A mitochondrial DNA mutation influences the apoptotic effect of statins on prostate cancer.

BACKGROUND: Statins, 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are currently the most widely used cholesterol-lowering drugs. Previous epidemiological studies have suggested that there may be be an association between statin use and decreased risk of prostate cancer progression. Both inherited and somatic mutations of the mitochondrial genome are linked to prostate cancer. The purpose of this study was to determine if mitochondrial DNA (mtDNA) background and hence mitochondrial biochemistry can modulate the efficiency of statin as an anti-prostate cancer agent. METHODS: Cytoplasmic hybrid (cybrid) cell lines were constructed that contained a prostate cancer nucleus and either wild type or mutant mtDNA derived from a prostate cancer patient with the cytochrome oxidase subunit 1 gene mutation T6124C (Met74Thr). Multiple clones for each genotype were tested. After treating both wild type and mutant cells with increasing concentrations of simvastatin for 72 hr, cell proliferation and apoptosis were analyzed. RESULTS: Simvastatin inhibited both wild type and mutant cell proliferation. However, cells with the T6124C mtDNA mutation were more resistant to drug treatment than the wild type cells. In addition, analysis of caspase 3 assays and multiple proteins involved in cellular apoptosis demonstrated that mutant cells were more resistant to simvastatin treatment-induced apoptosis than wild type control cells. CONCLUSIONS: Simvastatin treatment induced apoptosis in human cybrid prostate cancer cells. The response to drug treatments was different depending on mitochondrial genotype. Therefore, the degree to which statins may affect prostate cancer progression may vary based on an individual's mtDNA background.

Bone metastasis in prostate cancer: Recurring mitochondrial DNA mutation reveals selective pressure exerted by the bone microenvironment.

BACKGROUND: Cancer progression and metastasis occur such that cells with acquired mutations enhancing growth and survival (or inhibiting cell death) increase in number, a concept that has been recognized as analogous to Darwinian evolution of species since Peter C. Nowell's description in 1976. Selective forces include those intrinsic to the host (including metastatic site) as well as those resulting from anti-cancer therapies. By examining the mutational status of multiple tumor sites within an individual patient some insight may be gained into those genetic variants that enhance site-specific metastasis. By comparing these data across multiple individuals, recurrent patterns may identify alterations that are fundamental to successful site-specific metastasis. METHODS: We sequenced the mitochondrial genome in 10 prostate cancer patients with bone metastases enrolled in a rapid autopsy program. Patients had late stage disease and received androgen ablation and frequently other systemic therapies. For each of 9 patients, 4 separate tissues were sequenced: the primary prostate cancer, a soft tissue metastasis, a bone metastasis and an uninvolved normal tissue that served as the non-cancerous control. An additional (10th) patient had no primary prostate available for sequencing but had both metastatic sites (and control DNA) sequenced. We then examined the number and location of somatically acquired mitochondrial DNA (mtDNA) mutations in the primary tumor and two metastatic sites in each individual patient. Finally, we compared patients with each other to determine any common patterns of somatic mutation. RESULTS: Somatic mutations were significantly more numerous in the bone compared to either the primary tumor or soft tissue metastases. A missense mutation at nucleotide position (n.p.) 10398 (A10398G; Thr114Ala) in the respiratory complex I gene ND3 was the most common (7 of 10 patients) and was detected only in the bone. Other notable somatic mutations that occurred in more than one patient include a tRNA Arg mutation at n.p. 10436 and a tRNA Thr mutation at n.p. 15928. The tRNA Arg mutation was restricted to bone metastases and occurred in three of 10 patients (30%). Somatic mutation at 15928 was not restricted to the bone and also occurred in three patients. CONCLUSIONS: Mitochondrial genomic variation was greater in metastatic sites than in the primary tumor and bone metastases had statistically significantly greater numbers of somatic mutations than either the primary or the soft tissue metastases. The genome was not mutated randomly. At least one mutational "hot-spot" was identified at the individual base level (nucleotide position 10398 in bone metastases) indicating a pervasive selective pressure for bone metastatic cells that had acquired the 10398 mtDNA mutation. Two additional recurrent mutations (tRNA Arg and tRNA Thr) support the concept of bone site-specific "survival of the fittest" as revealed by variation in the mitochondrial genome and selective pressure exerted by the metastatic site.

Solenopsin A and analogs exhibit ceramide-like biological activity.

BACKGROUND: (-)-Solenopsin A is a piperidine alkaloid that is a component of the venom of the fire ant Solenopsis invicta. Previously, we have demonstrated that solenopsin exhibit anti-angiogenic activity and downregulate phosphoinositol-3 kinase (PI3K) in the p53 deficient renal cell carcinoma cell line 786-O. Solenopsin has structural similarities to ceramide, a major endogenous regulator of cell signaling and cancer therapy induced apoptosis. METHODS: Different analogs of solenopsin were synthesized in order to explore structure-activity relationships. The anti-proliferative effect of solenopsin and analogs was tested on six different cell lines, including three tumor cell lines, two normal cutaneous cell lines, and one immortalized hyperproliferative cell line. FRET-based reporters were used to study the affect of solenopsin and analogs on Akt activity and PDK1 activation and sucrose density gradient fractionation was performed to examine recruitment of PTEN to membrane rafts. Western-blotting was used to evaluate the affect of solenopsin and analogs on the Akt and the MAPK 44/42 pathways in three different tumor cell lines. Measurement of cellular oxygen consumption rate together with autophagy staining was performed to study mitochondrial function. Finally, the affect of solenopsin and analogs on ROS production was investigated. RESULTS: In this paper we demonstrate that solenopsin analogs with potent anti-proliferative effects can be synthesized from inexpensive dimethylpyridines. To determine whether solenopsin and analogs act as ceramide analogs, we examined the effect of solenopsin and analogs on two stereotypic sites of ceramide activity, namely at lipid rafts and mitochondria. We found that native solenopsin, (-)-solenopsin A, inhibits functional Akt activity and PDK1 activation in lipid rafts in a similar fashion as ceramide. Both cis and trans analogs of solenopsin reduce mitochondrial oxygen consumption, increase reactive oxygen, and kill tumor cells with elevated levels of Akt phosphorylation. However, only solenopsin induces mitophagy, like ceramide. CONCLUSIONS: The requirements for ceramide induced mitophagy and inhibition of Akt activity and PDK1 activation in lipid rafts are under strict stereochemical control. The naturally occurring (-)-solenopsin A mimic some of the functions of ceramide and may be therapeutically useful in the treatment of hyperproliferative and malignant disorders of the skin, even in the presence of elevated levels of Akt.

Mitochondrial DNA mutations in prostate cancer bone metastases.

Prostate cancer is the most common non-skin cancer in men. Those with local or regional disease often have good long-term prognosis, but patients with metastatic disease face high morbidity and mortality. The vast majority of cases with distant spread have some degree of bony involvement. The reason for the disproportionately high percentage of metastasis to bone relative to other metastatic sites remains unclear. A growing body of evidence suggests mitochondrial DNA (mtDNA) is associated with prostate cancer, and the effects of mtDNA on tumor growth may be augmented by the bone microenvironment. Here, we review our latest study analyzing mtDNA mutations in 10 patients with advanced prostate cancer and both bone and soft tissue metastases. This cohort of patients had significantly increased somatic mtDNA mutations in bone metastasis compared to paired primary tumor and soft tissue metastasis. In addition, a recurrent mtDNA mutation at nucleotide position 10398, was exclusively found in bone metastasis in 7 of 10 patients with advanced prostate cancer, with no such mutations found in paired benign prostate, primary tumor, or soft tissue metastasis. We describe the results from this work and review the relevant literature on the role of mitochondrial DNA in prostate cancer bone metastases.

Aquaporin-1 protein levels elevated in fresh urine of renal cell carcinoma patients: potential use for screening and classification of incidental renal lesions.

INTRODUCTION AND OBJECTIVES: There are over 65,000 new cases of renal cell carcinoma (RCC) each year, yet there is no effective clinical screening test for RCC. A single report claimed no overlap between urine levels of aquaporin-1 (AQP1) in patients with and without RCC (Mayo Clin Proc. 85:413, 2010). Here, we used archived and fresh RCC patient urine to validate this report. METHODS: Archived RCC, fresh prenephrectomy RCC, and non-RCC negative control urines were processed for Western blot analysis. Urinary creatinine concentrations were quantified by the Jaffe reaction (Nephron 16:31, 1976). Precipitated protein was dissolved in 1x SDS for a final concentration of 2 µg/µL creatinine. RESULTS: Negative control and archived RCC patient urine failed to show any AQP1 protein by Western blot analysis. Fresh RCC patient urine is robustly positive for AQP1. There was no signal overlap between fresh RCC and negative control, making differentiation straightforward. CONCLUSIONS: Our data confirms that fresh urine of patients with RCC contains easily detectable AQP1 protein. However, archival specimens showed an absence of detectable AQP1 indistinguishable from negative control. These findings suggest that a clinically applicable diagnostic test for AQP1 in fresh urine may be useful for detecting RCC.

von Hippel-Lindau exonic methylation analysis using MALDI-TOF mass spectrometry.

PURPOSE: Aberrant promoter methylation turns off gene expression and is involved in human malignancy. Studies show that first exon methylation has a tighter association with gene silencing compared to promoter methylation or gene mutation. However, to our knowledge the clinical importance of exonic methylation in renal cell carcinoma is unknown. We analyzed renal cell carcinoma for VHL gene exonic methylation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. MATERIALS AND METHODS: In 48 institutionally banked renal cell carcinoma patient tissue samples VHL exon sequencing was done as well as methylation analysis of promoter and exon 1 by mass spectrometry or conventional bisulfite analysis. Methylated human lymphocytic DNA (0% and 100%), nontemplate distilled H2O, and the UOK121 and UOK171 human renal cell carcinoma cell lines served as assay controls. Samples were considered hypermethylated if a CpG site showed greater than 50% methylation. RESULTS: Nine of the 43 patient samples read by our exon 1 assay had methylated VHL exon 1 sites, including 3 showing hypermethylation. The exon 1 methylation assay was robust and reproducible. Samples with exon 1 hypermethylation showed no exonic mutations. All samples assayed at VHL exon 2 were hypermethylated. CONCLUSIONS: To assay renal cell carcinoma tumors for VHL methylation matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is robust and reproducible, and capable of quantifying the methylation status of individual DNA bases. Exon 1 methylation may be an alternate mechanism of VHL gene silencing in renal cell carcinoma in addition to mutation and promoter methylation. Applying this assay in patient populations may allow enhanced diagnosis or tumor typing in the future.

Muscadine grape skin extract reverts snail-mediated epithelial mesenchymal transition via superoxide species in human prostate cancer cells.

BACKGROUND: Snail transcription factor can induce epithelial-mesenchymal transition (EMT), associated with decreased cell adhesion-associated molecules like E-cadherin, increased mesenchymal markers like vimentin, leading to increased motility, invasion and metastasis. Muscadine grape skin extract (MSKE) has been shown to inhibit prostate cancer cell growth and induce apoptosis without affecting normal prostate epithelial cells. We investigated novel molecular mechanisms by which Snail promotes EMT in prostate cancer cells via Reactive Oxygen Species (ROS) and whether it can be antagonized by MSKE. METHODS: ARCaP and LNCaP cells overexpressing Snail were utilized to examine levels of reactive oxygen species (ROS), specifically, superoxide, in vitro using Dihydroethidium (DHE) or HydroCy3 dyes. Mitosox staining was performed to determine whether the source of ROS was mitochondrial in origin. We also investigated the effect of Muscadine grape skin extract (MSKE) on EMT marker expression by western blot analysis. Migration and cell viability using MTS proliferation assay was performed following MSKE treatments. RESULTS: Snail overexpression in ARCaP and LNCaP cells was associated with increased concentration of mitochondrial superoxide, in vitro. Interestingly, MSKE decreased superoxide levels in ARCaP and LNCaP cells. Additionally, MSKE and Superoxide Dismutase (SOD) reverted EMT as evidenced by decreased vimentin levels and re-induction of E-cadherin expression in ARCaP-Snail cells after 3 days, concomitant with reduced cell migration. MSKE also decreased Stat-3 activity in ARCaP-Snail cells. CONCLUSIONS: This study shows that superoxide species may play a role in Snail transcription factor-mediated EMT. Therefore, therapeutic targeting of Snail with various antioxidants such as MSKE may prove beneficial in abrogating EMT and ROS-mediated tumor progression in human prostate cancer.

An inherited heteroplasmic mutation in mitochondrial gene COI in a patient with prostate cancer alters reactive oxygen, reactive nitrogen and proliferation.

Mitochondrial DNA (mtDNA) mutations have been found in many cancers but the physiological derangements caused by such mutations have remained elusive. Prostate cancer is associated with both inherited and somatic mutations in the cytochrome c oxidase (COI) gene. We present a prostate cancer patient-derived rare heteroplasmic mutation of this gene, part of mitochondrial respiratory complex IV. Functional studies indicate that this mutation leads to the simultaneous decrease in cytochrome oxidation, increase in reactive oxygen, and increased reactive nitrogen. These data suggest that mitochondrial DNA mutations resulting in increased reactive oxygen and reactive nitrogen generation may be involved in prostate cancer biology.

Antibody responses against xenotropic murine leukemia virus-related virus envelope in a murine model.

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) was recently discovered to be the first human gammaretrovirus that is associated with chronic fatigue syndrome and prostate cancer (PC). Although a mechanism for XMRV carcinogenesis is yet to be established, this virus belongs to the family of gammaretroviruses well known for their ability to induce cancer in the infected hosts. Since its original identification XMRV has been detected in several independent investigations; however, at this time significant controversy remains regarding reports of XMRV detection/prevalence in other cohorts and cell type/tissue distribution. The potential risk of human infection, coupled with the lack of knowledge about the basic biology of XMRV, warrants further research, including investigation of adaptive immune responses. To study immunogenicity in vivo, we vaccinated mice with a combination of recombinant vectors expressing codon-optimized sequences of XMRV gag and env genes and virus-like particles (VLP) that had the size and morphology of live infectious XMRV. RESULTS: Immunization elicited Env-specific binding and neutralizing antibodies (NAb) against XMRV in mice. The peak titers for ELISA-binding antibodies and NAb were 1:1024 and 1:464, respectively; however, high ELISA-binding and NAb titers were not sustained and persisted for less than three weeks after immunizations. CONCLUSIONS: Vaccine-induced XMRV Env antibody titers were transiently high, but their duration was short. The relatively rapid diminution in antibody levels may in part explain the differing prevalences reported for XMRV in various prostate cancer and chronic fatigue syndrome cohorts. The low level of immunogenicity observed in the present study may be characteristic of a natural XMRV infection in humans.

Snail-mediated regulation of reactive oxygen species in ARCaP human prostate cancer cells.

Reactive oxygen species increases in various diseases including cancer and has been associated with induction of epithelial-mesenchymal transition (EMT), as evidenced by decrease in cell adhesion-associated molecules like E-cadherin, and increase in mesenchymal markers like vimentin. We investigated the molecular mechanisms by which Snail transcription factor, an inducer of EMT, promotes tumor aggressiveness utilizing ARCaP prostate cancer cell line. An EMT model created by Snail overexpression in ARCaP cells was associated with decreased E-cadherin and increased vimentin. Moreover, Snail-expressing cells displayed increased concentration of reactive oxygen species (ROS), specifically, superoxide and hydrogen peroxide, in vitro and in vivo. Real Time PCR profiling demonstrated increased expression of oxidative stress-responsive genes, such as aldehyde oxidase I, in response to Snail. The ROS scavenger, N-acetyl cysteine partially reversed Snail-mediated EMT after 7 days characterized by increased E-cadherin levels and decreased ERK activity, while treatment with the MEK inhibitor, UO126, resulted in a more marked effect by 3 days, characterized by cells returning back to the epithelial morphology and increased E-cadherin. In conclusion, this study shows for the first time that Snail transcription factor can regulate oxidative stress enzymes and increase ROS-mediated EMT regulated in part by ERK activation. Therefore, Snail may be an attractive molecule for therapeutic targeting to prevent tumor progression in human prostate cancer.